http://2007.igem.org/wiki/index.php?title=Special:Contributions/Zhao_Yun&feed=atom&limit=50&target=Zhao_Yun&year=&month=2007.igem.org - User contributions [en]2024-03-29T09:01:41ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T05:28:01Z<p>Zhao Yun: /* Academic Activities */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systematic method.<br />
<br />
<br />
Here are the very rudiment of the three logic gates taken from one of our group meetings.<br />
<br />
{|<br />
|[[Image:ustc_nor gate.jpg|thumb|256px|First draft of our NOR gate]]<br />
|[[Image:ustc_nand gate.jpg|thumb|200px|First draft of our NAND gate]]<br />
|[[Image:ustc_not gate.jpg|thumb|200px|First draft of our NOT gate]]<br />
|}<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices, Jian ZHAN and Rui MA. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop in June 2007<br />
<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T05:27:03Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systematic method.<br />
<br />
<br />
Here are the very rudiment of the three logic gates taken from one of our group meetings.<br />
<br />
{|<br />
|[[Image:ustc_nor gate.jpg|thumb|256px|First draft of our NOR gate]]<br />
|[[Image:ustc_nand gate.jpg|thumb|200px|First draft of our NAND gate]]<br />
|[[Image:ustc_not gate.jpg|thumb|200px|First draft of our NOT gate]]<br />
|}<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices, Jian ZHAN and Rui MA. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop in June 2007<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T05:26:34Z<p>Zhao Yun: /* Research Work in iGEM */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systematic method.<br />
<br />
Here are the very rudiment of the three logic gates taken from one of our group meetings.<br />
<br />
{|<br />
|[[Image:ustc_nor gate.jpg|thumb|256px|First draft of our NOR gate]]<br />
|[[Image:ustc_nand gate.jpg|thumb|200px|First draft of our NAND gate]]<br />
|[[Image:ustc_not gate.jpg|thumb|200px|First draft of our NOT gate]]<br />
|}<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices, Jian ZHAN and Rui MA. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop in June 2007<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-27T02:17:03Z<p>Zhao Yun: /* Suggested Patterns */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 - 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
<br />
<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
<br />
<br />
<br />
----<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
----<br />
== Valuable Experiences for Bio-Logic Promoters ==<br />
<br />
=== Factors of Gate-Performances === <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|'''composition''']] and [[USTC/OperatorPosition|'''position''']].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|'''reported''']].<br />
<br />
=== Hybrid Operator and Dual-Repressed Operator ===<br />
Based on the data of [[USTC/OperatorComposition|'''operators' composition''']], two ideas have been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|'''Crossbreeding two specific operators''']] may be able to carry out new functions.<br />
* A specific operator repressed by two or more different repressors can be used as [[USTC/DualRepressedOperator|'''a model for NOR gate''']].<br />
<br />
<br />
<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|'''a uniform rule''']].<br />
<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is fixed at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. However, there is not any available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repression pattern works well as NOR gate, but it brings us a limitation in wires selection when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
The NOT gate is quite simple, containing only one operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]<br />
<br />
<br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-27T02:16:07Z<p>Zhao Yun: /* Suggested Patterns */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 - 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
<br />
<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
<br />
<br />
<br />
----<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
----<br />
== Valuable Experiences for Bio-Logic Promoters ==<br />
<br />
=== Factors of Gate-Performances === <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|'''composition''']] and [[USTC/OperatorPosition|'''position''']].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|'''reported''']].<br />
<br />
=== Hybrid Operator and Dual-Repressed Operator ===<br />
Based on the data of [[USTC/OperatorComposition|'''operators' composition''']], two ideas have been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|'''Crossbreeding two specific operators''']] may be able to carry out new functions.<br />
* A specific operator repressed by two or more different repressors can be used as [[USTC/DualRepressedOperator|'''a model for NOR gate''']].<br />
<br />
<br />
<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|'''a uniform rule''']].<br />
<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is fixed at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. However, there is not any available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selection when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
The NOT gate is quite simple, containing only one operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]<br />
<br />
<br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-27T02:14:33Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 - 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
<br />
<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
<br />
<br />
<br />
----<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
----<br />
== Valuable Experiences for Bio-Logic Promoters ==<br />
<br />
=== Factors of Gate-Performances === <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|'''composition''']] and [[USTC/OperatorPosition|'''position''']].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|'''reported''']].<br />
<br />
=== Hybrid Operator and Dual-Repressed Operator ===<br />
Based on the data of [[USTC/OperatorComposition|'''operators' composition''']], two ideas have been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|'''Crossbreeding two specific operators''']] may be able to carry out new functions.<br />
* A specific operator repressed by two or more different repressors can be used as [[USTC/DualRepressedOperator|'''a model for NOR gate''']].<br />
<br />
<br />
<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|'''a uniform rule''']].<br />
<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is fixed at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
The NOT gate is quite simple, containing only one operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]<br />
<br />
<br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-27T02:11:48Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 - 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
<br />
<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
<br />
<br />
<br />
----<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
----<br />
== Valuable Experiences for Bio-Logic Promoters ==<br />
<br />
=== Factors of Gate-Performances === <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|'''composition''']] and [[USTC/OperatorPosition|'''position''']].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|'''reported''']].<br />
<br />
=== Hybrid Operator and Dual-Repressed Operator ===<br />
Based on the data of [[USTC/OperatorComposition|'''operators' composition''']], two ideas have been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|'''Crossbreeding two specific operators''']] may be able to carry out new functions.<br />
* A specific operator repressed by two or more different repressors can be used as [[USTC/DualRepressedOperator|'''a model for NOR gate''']].<br />
<br />
<br />
<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|'''a uniform rule''']].<br />
<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
The NOT gate is quite simple, containing only one operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
<br />
<br />
<br />
----<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]<br />
<br />
<br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorCompositionUSTC/OperatorComposition2007-10-27T01:09:14Z<p>Zhao Yun: </p>
<hr />
<div>[[USTC/NamingRules|Naming Rules for Oxx and Oxy below]]<br />
<br />
<br />
<br />
Generally, Lac repressor bind to its operator in the form of dimer [[USTC/OperatorComposition#References|[1]]]. Almost all the according operators in nature are of approximate reverse symmetric structure, that is, each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer [[USTC/OperatorComposition#References|[2]]]. It has been reported that it is the perfectly symmetric operator that most tightly binds to native Lac repressor. Based on this fact, an idea was proposed that the binding affinity of a non-symmetric operator might be systematically reduced, exempt from the harm on the binding specificity of the repressor-operator pairs.<br />
<br />
<br />
Five promoters have been synthesized for testing the effect of weakening. Three of them are based on O11 operator:<br />
<br />
<br />
[[Image:USTC_AlignmentOfCompostions.png|frame|right]]<br />
* O11, the perfectly symmetric operator among these five ones<br />
* O1wt1, the native lac operator at +11 site of Plac promoter<br />
* O16, the right half of O11 replaced by the right half of O66<br />
* O17, the right half of O11 replaced by the right half of O77<br />
* NUL, the positive control as intensity 1.00<br />
([https://2007.igem.org/USTC/HybridOperator#Hybrid_Operators_for_Weaker_Binding more details about Ox6 and Ox7])<br />
<br />
<br />
<br />
[[Image:USTC_OperatorCompostions.png|thumb|center|512px|'''Figure 1''' The activating intensity of these promoters when corresponding specific repressor exsiting.]]<br />
<br />
<br />
After a series of our experiments (refer to Figure 1 whose data were from fluorescent measurement), we came to the conclusion that the method of replacing the right half of the symmetric operator can systematically reduce the binding affinity. Ox6, which means the right half of a symmetric operator is replaced by a weak operator sequence with the same pattern, can be used as a "weak" operator. At the same time, we found that the pattern Ox7 is weaker than that of Ox6, and [https://2007.igem.org/USTC/FailureOfNANDv2b the affinity of Ox7 is too low for a logic gate].<br />
<br />
<br />
Actually, we've made up O61, coming from O11 with its left half replaced by the left half of O66, but we found that it has no special feature contrast to O16 according to the data. For predigestion, we discard "O6x" series in the next works.<br />
<br />
<br />
<br />
<br />
== References ==<br />
<br />
1. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328 (6), 521--548.<br />
<br />
2. John R. Sadler, Henri Sasmor, and Joan L. Betz, A perfectly symmetric lac operator binds the lac repressor very tightly. <i>PNAS</i> Vol. 80, pp. 6785-6789, November 1983</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/HybridOperatorUSTC/HybridOperator2007-10-27T00:56:26Z<p>Zhao Yun: /* Hybrid Operators for Heterodimeric Repressor */</p>
<hr />
<div>[[USTC/NamingRules|Naming Rules for Oxx and Oxy below]]<br />
<br />
<br />
As we mentioned in [[USTC/OperatorComposition|'''Operator Composition''']], biologists always use symmetric operators like O22 because they are the most suitable ones to be bound to by their own specific repressor(s).<br />
<br />
==Hybrid Operators for Heterodimeric Repressor==<br />
Assumed that R4 is the highly specific repressor protein that well binds to O44, and so does R2 to O22, well, then it is clear that asymmetric O24 can be bound to both by R2 and R4. However, neither of them is very suitable because R4 is highly specific only for O44 rather than O22, and vice versa. Luckily these LacI-family Repressors are also symmetric, each of them is combined by two same monomers, so one side of R2 can bind to O24’s left-half strand while the other side would not bring about any remarkable effect on the right half. Binding to half strand could still maintain repression, but distinctly it could no longer be that strong. Now we can say that hybrid operator O24 is a “weaker” operator for R2 or R4 because their weaker binding compared with R2+O22 or R4+O44.<br />
<br />
[[Image:USTC_HybridOperator.png|500px|thumb|center|Supposed mechanism of hybrid operator. It appears as a weak operator to homodimers, and only heterodimers can bind to it tightly.]]<br />
<br />
==Hybrid Operators for Weaker Binding==<br />
There is another kind of “weaker” operators Ox6, for example, AATTGTGAAC GCTCATAATT (O46). The symmetric prototype AATTATGAGC GCTCATAATT (O66) is an interesting operator, because all the known LacI-family repressors would not have detectable repression on it [[USTC/HybridOperator#References|[1]]]. It is obvious that O46 is a “weaker” operator only to R4 (compared with R4+O44), but not a valid operator to other repressors that are not specific for O44. On the other hand, O24 is a “weaker” operator to both R4 and R2. We also try to use random sequence GACGACTGTA TACAGTCGTC (O77) to replace O66, that is to say, O47 is sequence AATTGTGAAC TACAGTCGTC. It seems that O47 might behave just like O46, but actually it is too weak to be bound to by R4 for our purpose. (Comparable data of Ox6 and Ox7 on page [[USTC/OperatorComposition|Operator Composition]].)<br />
<br />
== References ==<br />
<br />
1. Sartorius, J.; Lehming, N.; Kisters, B.; von Wilcken-Bergmann, B. & Müller-Hill, B. (1989), lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges., EMBO J 8(4), 1265--1270.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/HybridOperatorUSTC/HybridOperator2007-10-27T00:54:29Z<p>Zhao Yun: /* Hybrid Operators for Weaker Binding */</p>
<hr />
<div>[[USTC/NamingRules|Naming Rules for Oxx and Oxy below]]<br />
<br />
<br />
As we mentioned in [[USTC/OperatorComposition|'''Operator Composition''']], biologists always use symmetric operators like O22 because they are the most suitable ones to be bound to by their own specific repressor(s).<br />
<br />
==Hybrid Operators for Heterodimeric Repressor==<br />
Assumed that R4 is the highly specific repressor protein that well binds to O44, and so does R2 to O22, well then it is clear that asymmetric O24 can be bound to both by R2 and R4. However, neither of them is very suitable because R4 is highly specific only for O44 rather than O22, and vice versa. Luckily these LacI-family Repressors are also symmetric, each of them is combined by two same monomers, so one side of R2 can bind to O24’s left-half strand while the other side would not bring about any remarkable effect on the right. To bind to half strand could still maintain repression, but distinctly it could no longer be that strong. Now we can say that hybrid operator O24 is a “weaker” operator for R2 or R4 because their weaker binding compared with R2+O22 or R4+O44.<br />
<br />
[[Image:USTC_HybridOperator.png|500px|thumb|center|Supposed mechanism of hybrid operator. It appears as a weak operator to homodimers, and only heterodimers can bind to it tightly.]]<br />
<br />
<br />
<br />
==Hybrid Operators for Weaker Binding==<br />
There is another kind of “weaker” operators Ox6, for example, AATTGTGAAC GCTCATAATT (O46). The symmetric prototype AATTATGAGC GCTCATAATT (O66) is an interesting operator, because all the known LacI-family repressors would not have detectable repression on it [[USTC/HybridOperator#References|[1]]]. It is obvious that O46 is a “weaker” operator only to R4 (compared with R4+O44), but not a valid operator to other repressors that are not specific for O44. On the other hand, O24 is a “weaker” operator to both R4 and R2. We also try to use random sequence GACGACTGTA TACAGTCGTC (O77) to replace O66, that is to say, O47 is sequence AATTGTGAAC TACAGTCGTC. It seems that O47 might behave just like O46, but actually it is too weak to be bound to by R4 for our purpose. (Comparable data of Ox6 and Ox7 on page [[USTC/OperatorComposition|Operator Composition]].)<br />
<br />
== References ==<br />
<br />
1. Sartorius, J.; Lehming, N.; Kisters, B.; von Wilcken-Bergmann, B. & Müller-Hill, B. (1989), lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges., EMBO J 8(4), 1265--1270.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/NamingRulesUSTC/NamingRules2007-10-27T00:52:14Z<p>Zhao Yun: </p>
<hr />
<div>__NOTOC__<br />
<br />
Biologists always use symmetric LacI-family operators, e.g. AATTGTGAAC GTTCACAATT (O44) and AATTGTAAGC GCTTACAATT (O22). Please notice that they are all 20bp long and each right half of 10bp-long strand’s complementary strand is reversed symmetric with its left half of 10bp-long strand. For short we call this kinds of symmetrical operators as Oxx, for example, O44, O22, etc. Then you must have already understood what O24 means-- Yes, it means the asymmetric operator AATTGTAAGC GTTCACAATT (O24), which has the 10bp-long strand of O22 for its left half sequence and the 10bp-long one of O44 for its right half sequence. Again for example, O42 means AATTGTGAAC GCTTACAATT (O42), and so on. We also give these asymmetric operators another name: [[USTC/HybridOperator|Hybrid Operators]].<br />
<br />
<br />
For convenience, I listed the sequences we've used:<br />
<br />
<br />
<br />
===Symmetric Operators===<br />
<br />
O11 AATTGTGAGC GCTCACAATT<br />
O22 AATTGTAAGC GCTTACAATT<br />
O33 AATTGTAAAC GTTTACAATT<br />
O44 AATTGTGAAC GTTCACAATT<br />
O55 AATTTTGAGC GCTCAAAATT<br />
O66 AATTATGAGC GCTCATAATT<br />
O77 GACGACTGTA TACAGTCGTC<br />
<br />
<br />
===Asymmetric Operators===<br />
'''"Ox6", "Ox7" and "O6x"'''<br />
O16 AATTGTGAGC GCTCATAATT<br />
O26 AATTGTAAGC GCTCATAATT<br />
O46 AATTGTGAAC GCTCATAATT<br />
O61 AATTATGAGC GCTCACAATT<br />
O17 AATTGTGAGC TACAGTCGTC<br />
O27 AATTGTAAGC TACAGTCGTC<br />
O47 AATTGTGAAC TACAGTCGTC<br />
<br />
'''"Oxy"'''<br />
O12 AATTGTGAGC GCTTACAATT<br />
O21 AATTGTAAGC GCTCACAATT<br />
O24 AATTGTAAGC GTTCACAATT<br />
<br />
----<br />
<br />
===Those Operators===<br />
[[Image:USTC_Operators_D001Oxy.jpg|thumb|600px|left|]]<br />
<br style="clear:both;"><br />
<br />
<br />
===Loaded on Double Report System===<br />
[[Image:USTC_D01Oxy_DPv2GFP.jpg|thumb|600px|left|]]<br />
<br style="clear:both;"><br />
[[Image:USTC_D01Oxy_DPv2RFP.jpg|thumb|600px|left|]]<br />
<br style="clear:both;"><br />
<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorCompositionUSTC/OperatorComposition2007-10-27T00:47:29Z<p>Zhao Yun: </p>
<hr />
<div>[[USTC/NamingRules|Naming Rules for Oxx and Oxy below]]<br />
<br />
<br />
<br />
Generally, Lac repressor bind to its operator in the form of dimer [[USTC/OperatorComposition#References|[1]]]. Almost all the according operators in nature are of approximate reverse symmetric structure, that is, each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer [[USTC/OperatorComposition#References|[2]]]. It has been reported that it is the perfectly symmetric operator that most tightly binds to native Lac repressor. Based on this fact, an idea was proposed that the binding affinity of a non-symmetric operator might be systematically reduced, exempt from the harm on the binding specificity of the repressor-operator pairs.<br />
<br />
<br />
Five promoters have been synthesized for testing the effect of weakening. Three of them are based on O11 operator:<br />
<br />
<br />
[[Image:USTC_AlignmentOfCompostions.png|frame|right]]<br />
* O11, the perfectly symmetric operator in this five ones<br />
* O1wt1, the native lac operator at +11 site of Plac promoter<br />
* O16, the right half of O11 replaced by the right half of O66<br />
* O17, the right half of O11 replaced by the right half of O77<br />
* NUL, the positive control as intensity 1.00<br />
([https://2007.igem.org/USTC/HybridOperator#Hybrid_Operators_for_Weaker_Binding more details about Ox6 and Ox7])<br />
<br />
<br />
<br />
[[Image:USTC_OperatorCompostions.png|thumb|center|512px|'''Figure 1''' The activating intensity of these promoters when corresponding specific repressor exsiting.]]<br />
<br />
<br />
After a series of our experiments (refer to Figure 1 whose data were from fluorescent measurement), we came to the conclusion that the method of replacing the right half of the symmetric operator can systematically reduce the binding affinity. Ox6, which means the right half of a symmetric operator is replaced by a weak operator sequence with the same pattern, can be used as a "weak" operator. At the same time, we found that the pattern Ox7 is weaker than that of Ox6, and [https://2007.igem.org/USTC/FailureOfNANDv2b the affinity of Ox7 is too low for a logic gate].<br />
<br />
<br />
Actually, we've made up O61, coming from O11 with its left half replaced by the left half of O66, but we found that it has no special feature contrast to O16 according to the data. For predigestion, we discard "O6x" series in the next works.<br />
<br />
<br />
<br />
<br />
== References ==<br />
<br />
1. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328 (6), 521--548.<br />
<br />
2. John R. Sadler, Henri Sasmor, and Joan L. Betz, A perfectly symmetric lac operator binds the lac repressor very tightly. <i>PNAS</i> Vol. 80, pp. 6785-6789, November 1983</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/FailureOfNANDv2bUSTC/FailureOfNANDv2b2007-10-27T00:44:02Z<p>Zhao Yun: </p>
<hr />
<div>NANDv2b has not passed the X-gal assay on plates, so no ONPG data has been measured. This is caused by the weakest hybrid operator downstream. The affinity of this operator (O27) is too weak for repressor to bind to the DNA and repress the promoter.<br />
<br />
[[Image:USTC_NANDv2b_Xgal.jpg|thumb|500px|center|'''Figure 1''' X-gal plates of NANDv2b. From left to right: R2+R4, R2, R4. Blue colonies show that the weakest hybrid operator is not suitable for NAND gate.]]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:35:24Z<p>Zhao Yun: /* Research Work in iGEM */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices, Jian ZHAN and Rui MA. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop in June 2007<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:33:22Z<p>Zhao Yun: /* Academic Activities */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop in June 2007<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:30:04Z<p>Zhao Yun: /* Research Work in iGEM */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]].<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:29:28Z<p>Zhao Yun: /* Research Work in iGEM */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to [[USTC/Logic-Gate_Promoters|Logic-Gate Promoters]]<br />
<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:28:57Z<p>Zhao Yun: /* Research Work in iGEM */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to ----<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:28:36Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to ----<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-27T00:23:15Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
[[Image:Example.jpg]]<br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to ----<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorPositionUSTC/OperatorPosition2007-10-26T12:53:34Z<p>Zhao Yun: </p>
<hr />
<div> <br />
Different locations of an operator plays a role in determining the intensity of repression in vivo[[USTC/OperatorPosition#References|[1]]].Both operators with strong and weak repression are required to the gates. Therefore, we systematically tested the influence of an operator at different locations on the promoter activity.<br />
<br />
Downstream Operaters exhibit intense repression effect when bound by their according repressors. The closer they are fixed to the consensus sequence, the intenser the repression will be. Repression resulted from faraway repressor-operator pairs is so weak that it could be ignored in some situation. In contrast, repressor-operator pairs upstream of the consensus sequence have more or less shown the activation effect rather than repressed the promter activity, which made us much astonished. (Please refer to Figure 1 and 2)<br />
[[Image:USTC_RelativePromoterActivity.png|thumb|600px|center|'''Figure 1''' Left: the influence on the promoter’s activity when a single operator is at a different position upstream of the consensus sequence. <br />
Right: the influence on the promoter’s activity when a single operator is at a different position downstream of the consensus sequence.<br />
]]<br />
<br />
[[Image:USTC_EffectOfPosition.png|thumb|600px|center|'''Figure 2''' Assumed influence on solo-repression when an operator is at a different position.]]<br />
<br />
== References ==<br />
<br />
1. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T12:36:38Z<p>Zhao Yun: /* Suggested Patterns */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
<br />
==== Factors of Gate-Performances ==== <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|composition]] and [[USTC/OperatorPosition|position]].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|reported]].<br />
<br />
==== Hybrid Operator and Dual-Repressed Operator ====<br />
Based on the data of [[USTC/OperatorComposition|operators' composition]], two ideas has been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|Crossbreeding two specific operators]] may carry out new functions<br />
* A specific operator repressed by two different repressors can be used as [[USTC/DualRepressedOperator|a model for our NOR gate]].<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|a uniform rule]].<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
The NOT gate is quite simple, containing only one operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/DualRepressedOperatorUSTC/DualRepressedOperator2007-10-26T12:33:50Z<p>Zhao Yun: </p>
<hr />
<div>{|<br />
| [[Image:USTC_DualRepressedOperator.png|thumb|512px|'''Figure 1''' Sketch map of Dual-Repressed Operator.]]<br />
| [[Image:USTC_BestNOR.png|thumb|256px|'''Figure 2''' Best NOR, an example of Dual-Repressed Operators.]]<br />
|}<br />
<br />
<br />
As shown in Figure 1 and 2, promoter with a so-called "Dual-Repressed Operator" in the downstream can be used as a well-performing NOR gate. However, we faced a dilemma when integrating these dual-repressed NOR Gates into an actual system. It was because that most of the components in the system require wires exempt from interference while these NOR gates just in the contrary manner take the advantage of the mentioned interference. Therefore, the Dual-Repressed Operator should be carefully selected from the Repression Matrix (Figure 3). ([[USTC/Repressor_Evolution_on_Plates|more details about this Matrix]])<br />
<br />
<br />
<br />
[[Image:USTC_RepressionMatrix.png|thumb|center|450px|'''Figure 3''' Repression Matrix]]<br />
<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorPositionUSTC/OperatorPosition2007-10-26T12:30:31Z<p>Zhao Yun: </p>
<hr />
<div> <br />
Different locations of an operator plays a role in determining the intensity of repression in vivo[[USTC/OperatorPosition#References|[1]]].Both operators with strong and weak repression are required to the gates. Therefore, we systematically tested the influence of an operator at different locations on the promoter activity.<br />
[[Image:USTC_RelativePromoterActivity.png|thumb|600px|center|'''Figure 1''' Left: the influence on the promoter’s activity when a single operator is at a different position upstream of the consensus sequence. <br />
Right: the influence on the promoter’s activity when a single operator is at a different position downstream of the consensus sequence.<br />
]]<br />
<br />
[[Image:USTC_EffectOfPosition.png|thumb|600px|center|'''Figure 2''' Assumed influence on solo-repression when an operator is at a different position.]]<br />
<br />
== References ==<br />
<br />
1. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorPositionUSTC/OperatorPosition2007-10-26T12:28:39Z<p>Zhao Yun: </p>
<hr />
<div> <br />
Different locations of an operator plays a role in determining the intensity of repression in vivo[[USTC/OperatorPosition#References|[1]]].Both operators with strong and weak repression are required to the gates. Therefore, we systematically tested the influence of operators at different locations on the promoter activity.<br />
[[Image:USTC_RelativePromoterActivity.png|thumb|600px|center|'''Figure 1''' Left: the influence on the promoter’s activity when a single operator is at a different position upstream of the consensus sequence. <br />
Right: the influence on the promoter’s activity when a single operator is at a different position downstream of the consensus sequence.<br />
]]<br />
<br />
[[Image:USTC_EffectOfPosition.png|thumb|600px|center|'''Figure 2''' Assumed influence on solo-repression when an operator is at a different position.]]<br />
<br />
== References ==<br />
<br />
1. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorPositionUSTC/OperatorPosition2007-10-26T12:27:37Z<p>Zhao Yun: </p>
<hr />
<div> <br />
Different locations of an operator plays a role in determining the intensity of repression in vivo[[USTC/OperatorPosition#References|[1]]].Both operators with strong and weak repression are required to the gates. Therefore, we systematically tested the influence of location factor on the promoter activity.<br />
[[Image:USTC_RelativePromoterActivity.png|thumb|600px|center|'''Figure 1''' Left: the influence on the promoter’s activity when a single operator is at a different position upstream of the consensus sequence. <br />
Right: the influence on the promoter’s activity when a single operator is at a different position downstream of the consensus sequence.<br />
]]<br />
<br />
[[Image:USTC_EffectOfPosition.png|thumb|600px|center|'''Figure 2''' Assumed influence on solo-repression when an operator is at a different position.]]<br />
<br />
== References ==<br />
<br />
1. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/OperatorCompositionUSTC/OperatorComposition2007-10-26T12:14:58Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:USTC_OperatorCompostions.png|thumb|right|400px]]<br />
<br />
Generally, Lac repressor bind to its operator in the form of dimer. Almost all the according operators in nature are of approximate reverse symmetric structure, that is, each half of the operators consists of about 10 nucleotides and receives the binding of a repressor monomer. It has been reported that it is the perfectly symmetric operator that most tightly binds to native Lac repressor. Based on this fact, an idea was proposed that the binding affinity of a non-symmetric operator might be systematically reduced, exempt from the harm on the binding specificity of the repressor-operator pairs.<br />
<br />
Five promoters have been synthesized and tested:<br />
* O11, the perfectly symmetric operator<br />
* O1wt1, the native lac operator at +11 site of Plac promoter<br />
* O16, the right half of O11 replaced by a sequence that weakly binds to Lac repressor, but has the same pattern as O11's.<br />
* O17, the right half of O11 replaced by a random sequence<br />
* NUL, the positive control<br />
<br />
[[Image:USTC_AlignmentOfCompostions.png|frame|center]]<br />
<br />
After a series of experiments, we came to the conclusion that the method of replacing the right half of the symmetric operator can systematically reduce the binding affinity. Ox6, which means the right half of a symmetric operator is replaced by a weak operator sequence with the same pattern, can be used as a "weak" operator. At the same time, we found that the pattern Ox7 is weaker than that of Ox6, and [https://2007.igem.org/USTC/FailureOfNANDv2b the affinity of Ox7 is too low for a logic gate].</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T12:06:33Z<p>Zhao Yun: /* Hybrid Operator and Dual-Repressed Operator */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
<br />
==== Factors of Gate-Performances ==== <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|composition]] and [[USTC/OperatorPosition|position]].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|reported]].<br />
<br />
==== Hybrid Operator and Dual-Repressed Operator ====<br />
Based on the data of [[USTC/OperatorComposition]], two ideas has been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|Crossbreeding two specific operators]] may carry out new functions<br />
* A specific operator repressed by two different repressors can be used as [[USTC/DualRepressedOperator|a model for our NOR gate]].<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|a uniform rule]].<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T12:04:46Z<p>Zhao Yun: /* Hybrid Operator and Dual-Repressed Operator */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
<br />
==== Factors of Gate-Performances ==== <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|composition]] and [[USTC/OperatorPosition|position]].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|reported]].<br />
<br />
==== Hybrid Operator and Dual-Repressed Operator ====<br />
Based on the data of [[USTC/OperatorComposition]], two ideas has been proposed and attempted to realize:<br />
* [[USTC/HybridOperator|Crossbreeding two specific operators]] may carry out new functions<br />
* A specific operator repressed by two different repressors can be used as [[USTC/DualRepressedOperator|the essential element for our NOR gate]].<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
All the members of the our promoter family are named according to [[USTC/NamingRules|a uniform rule]].<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T08:23:02Z<p>Zhao Yun: /* Systematical measure of repression's level */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
<br />
==== Systematical measure of repression's level ==== <br />
The level of repression in vivo is determined by several factors . Because both 'strong' operator and 'weak' operator are required for our system, we systematically tested the effects by the two primary factors: [[USTC/OperatorComposition|composition]] and [[USTC/OperatorPosition|position]].The proper distance between two operators is also necessary for NOR and NAND gates, and data has been [[USTC/InterOperatorDistance|systematically measured]].<br />
<br />
==== Heterodimer gates ====<br />
Besides the gates based on homodimers, [[USTC/HybridOperator|NAND]] and [[USTC/CoRepressedOperator|NOR]] gates based on heterodimers are also attempted .<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Further_MoreUSTC/Further More2007-10-26T08:10:17Z<p>Zhao Yun: </p>
<hr />
<div>== Further Optimization ==<br />
<br />
Though the existing bio-logic gates and wires are able to form a practicable combinational logic circuit in a bacterial cell, there remain some bottlenecks limiting the capability of our method. <br />
<br />
* '''Size of the Wires'''<br>The size of wires are much larger than the gates in our system. We think it should be reduced. Some ligands, for example, peptides, saccharides, lipid and so on, might be used as signal carrier.<br />
<br />
* '''More Input Signals'''<br>More signals with high quality are needed in larger-scale bio-logic circuit to serve as inputs. We think computational protein-ligand design can play an important role in providing more highly-specificied protein-ligand pairs.<br />
<br />
* '''Response Time'''<br>At present, the response time of our bio-logic gates is much longer than that of electronic ones. This situation should be improved. <br />
<br />
* '''I/O Standardization'''<br>Intensity of the input and output signals for different gates should be adjusted to the same level. There are several proposals as following.<br />
<br />
<br />
<br />
== Conductance Adjusting ==<br />
<br />
As mentioned above, the non-interference Repressor/Operator pairs can function well as “wires”. Of course, different pairs have different strength of bond. Binding intensity may also be different even if the two components are of the same type. Therefore, these wires will transmit different intensity of signals from upstream components to downstream ones. In qualitative or semiquantitative experiments, this will not be a serious problem, but we must solve it for experiments that are more precise.<br />
<br />
<br />
We all know that different promoters can initiate transcription strongly or weakly ([http://partsregistry.org/Part:BBa_J23100 Parts:BBa_J23100 and its family]). On the other hand, various RBS may lead to various efficiency of translation ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). Naturally, we will think of the following two approaches: using different promoters or using different RBS. However, according to the idea of “modularization”, we don’t want to casually modify promoters because these promoters are the kernel elements of our logic components. Anyway, we can modify other elements around this kernel, i.e. the RBS and operators. Let’s imagine the wires--every wire has two ends, and each of our Repressor/Operator pairs is a monodirectional wire. The beginning end is the RBS that activates the translation of this repressor protein, and the tail end is the operator to which an according repressor can bind. <br />
<br />
<br />
To modify the RBS or operators is just like changing the conductance at the junctions of actual wires, so we call it “Conductance Adjusting”:<br />
<br />
===Using different RBS===<br />
<br />
Registry of Standard Biological Parts have provided many kinds of RBS whose activities varies in a wide range ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). We’ve submitted our repressor protein coding parts without RBS or terminator so that every one can assemble them with appropriate RBS to get any efficiency of translation he wants.<br />
<br />
[[Image:USTC NoRBS.png]]<br />
<br />
<br />
We have also submitted those protein coding parts with RBS B0034 and terminator B0015 assembled. If you care nothing for the difference between different RBS, well, then they are good enough.<br />
<br />
[[Image:USTC WithRBS.png]]<br />
<br />
<br />
===Using different Operators===<br />
<br />
''TODO by Ma Rui''<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Further_MoreUSTC/Further More2007-10-26T08:08:46Z<p>Zhao Yun: </p>
<hr />
<div>== Further Optimization ==<br />
<br />
Though the existing bio-logic gates and wires are able to form a practicable combinational logic circuit in a bacterial cell, there remain some bottlenecks limiting the capability of our method. <br />
<br />
* '''Size of the Wires'''<br>The size of wires are much larger than the gates in our system. We think it should be reduced. Some ligands, for example, peptides, saccharides, lipid and so on, might be used as signal carrier.<br />
<br />
* '''More Input Signals'''<br>More signals with high quality are needed in larger-scale bio-logic circuit to serve as inputs. We think computational protein-ligand design can play an important role in providing more highly-specificied protein-ligand pairs.<br />
<br />
* '''Response Time'''<br>At present, the response time of our bio-logic gates is much longer than that of electronic ones. This situation should be improved. <br />
<br />
* '''I/O Standardization'''<br>Intensity of the input and output signals for different gates should be adjusted to the same level. There are several proposals as following.<br />
<br />
<br />
<br />
== Conductance Adjusting ==<br />
<br />
As mentioned above, the non-interference Repressor/Operator pairs can function well as “wires”. Of course, different pairs have different strength of bond. Binding intensity may also be different even if the two components are of the same type. Therefore, these wires will transmit different intensity of signals from upstream components to downstream ones. In qualitative or semiquantitative experiments, this will not be a serious problem, but we must solve it for experiments that are more precise.<br />
<br />
<br />
We all know that different promoters can initiate transcription strongly or weakly ([http://partsregistry.org/Part:BBa_J23100 Parts:BBa_J23100 and its family]). On the other hand, various RBS may lead to various efficiency of translation ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). Naturally, we will think of the following two approaches: using different promoters or using different RBS. However, according to the idea of “modularization”, we don’t want to casually modify promoters because these promoters are the kernel elements of our logic components. Anyway, we can modify other elements around this kernel, i.e. the RBS and operators. Let’s imagine the wires--every wire has two ends, and each of our Repressor/Operator pairs is a monodirectional wire. The beginning end is the RBS that activates the translation of this repressor protein, and the tail end is the operator to which an according repressor can bind. <br />
<br />
<br />
To modify the RBS or operators is just like changing the conductance of actual wires’ junctions''', so we call it “Conductance Adjusting”:<br />
<br />
===Using different RBS===<br />
<br />
Registry of Standard Biological Parts have provided many kinds of RBS whose activities varies in a wide range ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). We’ve submitted our repressor protein coding parts without RBS or terminator so that every one can assemble them with appropriate RBS to get any efficiency of translation he wants.<br />
<br />
[[Image:USTC NoRBS.png]]<br />
<br />
<br />
We have also submitted those protein coding parts with RBS B0034 and terminator B0015 assembled. If you care nothing for the difference between different RBS, well, then they are good enough.<br />
<br />
[[Image:USTC WithRBS.png]]<br />
<br />
<br />
===Using different Operators===<br />
<br />
''TODO by Ma Rui''<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Further_MoreUSTC/Further More2007-10-26T07:51:53Z<p>Zhao Yun: </p>
<hr />
<div>== Further Optimization ==<br />
<br />
Though the existing bio-logic gates and wires are able to form a practicable combinational logic circuit in a bacterial cell, there remain some bottlenecks limiting the capability of our method. <br />
<br />
* '''Size of the Wires'''<br>The size of wires are much larger than the gates in our system. We think it should be reduced. Some ligands, for example, peptides, saccharides, lipid and so on, might be used as signal carrier.<br />
<br />
* '''More Input Signals'''<br>More signals with high quality are needed in larger-scale bio-logic circuit to serve as inputs. We think computational protein-ligand design can play an important role in providing more highly-specificied protein-ligand pairs.<br />
<br />
* '''Response Time'''<br>At present, the response time of our bio-logic gates is much longer than that of electronic ones. This situation should be improved. <br />
<br />
* '''I/O Standardization'''<br>Intensity of the input and output signals for different gates should be adjusted to the same level. There are several proposals as following.<br />
<br />
<br />
<br />
== Conductance Adjusting ==<br />
<br />
As mentioned above, the non-interference Repressor/Operator pairs can function well as “wires”. Of course, different pairs have different strength of bond, so these wires will transmit different intensity of signals from upstream components to downstream ones, '''even though conducting the same two components.''' In qualitative or semiquantitative experiments, this will not be a serious problem, but we must solve it for experiments that are more precise.<br />
<br />
<br />
We all know that different promoters can initiate transcription strongly or weakly ([http://partsregistry.org/Part:BBa_J23100 Parts:BBa_J23100 and its family]). On the other hand, various RBS may lead to various efficiency of translation ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). Naturally, we will think of the following two approaches: using different promoters or using different RBS. However, according to the idea of “modularization”, we don’t want to casually modify promoters because these promoters are the kernel elements of our logic components. Anyway, we can modify other elements around this kernel, i.e. the RBS and operators. Let’s imagine the wires--every wire has two ends, and each of our Repressor/Operator pairs is a monodirectional wire. The beginning terminal is the RBS that activates the translation of this repressor protein, and the ending terminal is the operator that [[accesses]] the repression of this protein. <br />
<br />
<br />
To modify the RBS or operators is just like changing''' the conductance of actual wires’ ends''', so we call it “Conductance Adjusting”:<br />
<br />
===Using different RBS===<br />
<br />
Registry of Standard Biological Parts have provided many kinds of RBS whose activities '''distributes''' in a wide range ([http://partsregistry.org/Part:BBa_J61100 Parts:BBa_J61100 and its family] and [http://partsregistry.org/JCA_Arkin_RBSFamilyPart2 JCA_Arkin_RBS Family]). We’ve submitted our repressor protein coding parts without RBS or terminator so that every one can assemble them with appropriate RBS to get '''applicable efficiency of translation'''.<br />
<br />
[[Image:USTC NoRBS.png]]<br />
<br />
<br />
We have also submitted those protein coding parts with RBS B0034 and terminator B0015 assembled. If you care nothing for the difference between different RBS, well, then they are good enough.<br />
<br />
[[Image:USTC WithRBS.png]]<br />
<br />
<br />
===Using different Operators===<br />
<br />
''TODO by Ma Rui''<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/DemonstrationUSTC/Demonstration2007-10-26T07:18:36Z<p>Zhao Yun: </p>
<hr />
<div>An actual demonstration is determined to show the extensibility of our method. <br />
<br />
<br />
This demo system<br />
* is designed as simple as possible, without any "cool" logic function; <br />
* includes all the three logic gates, which constitute a three-level logic circuit; <br />
* shows that wires can cross and branch off (without interference); <br />
* is loaded on two plasmids, [[http://partsregistry.org/Part:BBa_I732923 pSB1A3-I732923]] and [[http://partsregistry.org/Part:BBa_I732925 pSB3K5-I732925]] ,and practically transformed into Top10 strain; <br />
<br />
* accepts aTc and AHL signals as inputs; <br />
* produces RFP and GFP signals as outputs; <br />
* can be "reset" by IPTG signal (i.e. both of the outputs are lightened when IPTG added in); <br />
* is expected to put out the results that accord with the truth value shown in Figure 2. <br />
<br />
<br />
=Logic Abstract of the Demo=<br />
<br />
[[Image:DemonstrationLogic.png|thumb|512px|left|'''Figure 1''' The logic diagram of the demo system.]]<br />
<br />
<br style="clear:both;"><br />
<br />
[[Image:DemonstrationTruthTable.png|thumb|320px|left|'''Figure 2''' The truth table of the demo system.]]<br />
<br style="clear:both;"><br />
<br />
We can see from this truth table that IPTG might result in both of the outputs being lightened no matter whether aTc or AHL exists or not. (It is just like the response "8888..." on the screen when you reset some of your electrical apparatus, for example, a calculator.)<br />
<br />
=Actual System=<br />
[[Image:DemonstrationSystem.png|thumb|512px|left|'''Figure 3''' The signal pathway of the demo system.]]<br />
<br style="clear:both;"><br />
<br />
The three parts on the left are loaded on [[http://partsregistry.org/Part:BBa_I732923 pSB1A3-I732923]], and the rest two parts are loaded on [[http://partsregistry.org/Part:BBa_I732925 pSB3K5-I732925]]. We've built up this system in one kind of TOP10 ''E.coli'', but it seems that it cannot grow stably when concentrated aTc exists. (We have to use aTc at a high concentration for counteracting the high expression of LuxR, but concentrated aTc shows some bacteriostatic activity like Tc(TetraCycline).) We may not be able to send out the very final results here exactly by Oct. 26, but we have continually been trying our best to screen out such a monoclonal strain that is not influenced by concentrated aTc. You will get the results in the coming presentation...<br />
<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/DemonstrationUSTC/Demonstration2007-10-26T07:14:24Z<p>Zhao Yun: </p>
<hr />
<div>An actual demonstration is determined to show the extensibility of our method. <br />
<br />
<br />
This demo system<br />
* is designed as simple as possible, without any "cool" logic function; <br />
* includes all the three logic gates, which constitute a three-level logic circuit; <br />
* shows that wires can cross and branch off (without interference); <br />
* is loaded on two plasmids, [[http://partsregistry.org/Part:BBa_I732923 pSB1A3-I732923]] and [[http://partsregistry.org/Part:BBa_I732925 pSB3K5-I732925]] ,and practically transformed into Top10 strain; <br />
<br />
* accepts aTc and AHL signals as inputs; <br />
* produces RFP and GFP signals as outputs; <br />
* can be "reset" by IPTG signal (i.e. both of the outputs are lightened when IPTG added in); <br />
* is expected to put out the results that accord with the truth value shown in Figure 2. <br />
<br />
<br />
=Logic Abstract of the Demo=<br />
<br />
[[Image:DemonstrationLogic.png|thumb|512px|left|'''Figure 1''' The logic diagram of the demo system.]]<br />
<br />
<br style="clear:both;"><br />
<br />
[[Image:DemonstrationTruthTable.png|thumb|320px|left|'''Figure 2''' The truth table of the demo system.]]<br />
<br style="clear:both;"><br />
<br />
We can see from this truth table that IPTG might result in both of the outputs being lightened no matter whether aTc or AHL exists or not. (It is just like the response "8888..." on the screen when you reset one of your digital equipment, for example, a calculator.)<br />
<br />
=Actual System=<br />
[[Image:DemonstrationSystem.png|thumb|512px|left|'''Figure 3''' The signal pathway of the demo system.]]<br />
<br style="clear:both;"><br />
<br />
The three parts on the left are loaded on [[http://partsregistry.org/Part:BBa_I732923 pSB1A3-I732923]], and the rest two parts are loaded on [[http://partsregistry.org/Part:BBa_I732925 pSB3K5-I732925]]. We've built up this system in one kind of TOP10 ''E.coli'', but it seems that it cannot grow stably when concentrated aTc exists. (We have to use aTc at a high concentration for counteracting the high expression of LuxR, but concentrated aTc shows some bacteriostatic activity like Tc(TetraCycline).) We may not be able to send out the very final results here exactly by Oct. 26, but we have continually been trying our best to screen out such a monoclonal strain that do not care about concentrated aTc. You will get the results in the coming presentation...<br />
<br />
<br />
<br><br />
<br></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Inputs_and_OutputsUSTC/Inputs and Outputs2007-10-26T07:10:32Z<p>Zhao Yun: </p>
<hr />
<div>= Input Devices =<br />
<br />
PoPS (the flow of RNA Polymerase molecules along DNA) means the current of gene expression. Our logic gates accept PoPS input signals, but we cannot input a PoPS signal directly. We decide to add something into our system as input signal, such as cheminal ligands and light. So a convertor must be built firstly to convert these signals to PoPS signals.<br />
<br />
<br />
<br />
== Candidates of input signals ==<br />
=== Light ===<br />
We decide not to use light signals as the inputs of our system for the following two reasons. <br />
<br />
For one, it would be rather difficult to dynamically control the light intensity in real experiments. Compared with simply adding reagents to the solutions, controlling the input level of light is far more complicated. Still unpleasant is the requirement for the techniques of keeping the light sensors on the cell membrane from frequent exposure. <br />
<br />
For another, since light has seldom been applied as input signals before, there is little relative background knowledge and experience to help us.<br />
<br />
=== IPTG ===<br />
<br />
IPTG is actually the RESET signal of our whole system. Once added, it will let all the repressor-operator pairs in the system no longer function. All the outputs will consequently become 1 as well as all the input repressing signals being automatically ignored. (It just likes the "8888..." on the screen when you reset your calculator or some other digital equipment.)<br />
<br />
=== aTc ===<br />
<br />
ATC is a well studied and widely used inducer of cellular communication. We take a fancy to its stable properties and reasonable price. Certainly, there are still some problems, for example, aTc at a high concentration shows some bacteriostatic activity like Tc(TetraCycline). Unfortunately we have to use concentrated aTc for counteracting the high expression of TetR repressor protein.<br />
<br />
=== AHL ===<br />
<br />
We consider AHL a well-qualified input signal that is normally used in quorum sensing. Actually, we choose 3OC6HSL, a member of AHL family to be the input signal, and its according receptor LuxR to be the sensor. Here we find it no problem to use dilute AHL although LuxR is also high expressing, because LuxR is activator and AHL is its accelerator.<br />
<br />
=== Arabinose ===<br />
<br />
Considering that the promoter of arabinose requires to match and is too much limited by the genotype of the host strain, we finally give up the idea that arabinose would serve as an input signal.<br />
<br />
<br />
----<br />
<br />
== Design of PoPS converters ==<br />
<br />
[[Image:PoPS Converters.jpg|386px|left|'''Figure 1''' Design of PoPS converters.]]<br />
<br style="clear:both;"><br />
<br />
The figure above shows the basic structure of our input device. It functions to convert the input chemical signals to the PoPS (the flow of RNA Polymerase molecules along DNA) signals of the lac repressor A and B, the wires that will actually connect the two main parts of our system together.<br />
<br />
== PoPS-converter parts ==<br />
<br />
[[Image:ustc_atc_pops.jpg|thumb|right|192px|left|'''Figure 2''' The first design of [aTc]->PoPS converter (BBa_I732014).]]<br />
<br style="clear:both;"><br />
<br />
Figure 2 is the first design of our [aTc]->PoPS converter. Fluorescent reporters was added behind. However, we found that the background expression could not be ignored under the fluorescent microscope. It might be due to the LVA tag of Tet repressor which served to help TetR degrade. Therefore, we decide to remove the LVA tag of the Tet repressor like the one shown in Figure 3.<br />
<br />
We take [http://partsregistry.org/Part:BBa_J09855 BBa_J09855] directly from the registry as [AHL]->PoPS converter. Fluorescent expression under different [3OC6HSL] has been tested and the results show that this part is a perfect input device in this study.<br />
<br />
[[Image:USTC_aTc_PoPS_2.jpg|thumb|right|192px|left|'''Figure 3''' The second design of [aTc]->PoPS converter (BBa_I732083).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:USTC_AHL_PoPS.jpg|thumb|right|192px|left|'''Figure 4''' [AHL]->PoPS converter (BBa_J09855).]]<br />
<br style="clear:both;"><br />
<br />
= Output Devices =<br />
For the sake of convenience, we choose LacZ and fluorescent proteins as the corresponding qualitative and quantitative reporters. With LacZ, we can roughly estimate the property of a gate candidate while with fluorescent proteins, we can further examine with a fluorescent microscope candidates that has passed the LacZ test, and finally pick out the best ones.<br />
<br />
== Fluorescent Proteins ==<br />
Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) are used in our system as reporters. In most of the measurements, we use the stable version ([http://partsregistry.org/Part:BBa_E0040 BBa_E0040] and [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]). But these fluorescent proteins are too stable for fast degradation. Therefore, the unstable version with LVA tag have also been synthesized ([http://partsregistry.org/Part:BBa_I732077 BBa_I732077] and [http://partsregistry.org/Part:BBa_I732078 BBa_I732078]) to reduce the half life. However, the untagged fluorescent proteins could better approach our expectation and requirements, so we construct the final version of our reporter system with the stable RFP and GFP.<br />
<br />
== LacZ: Beta-Galactosidase Activity ==<br />
LacZ is widely used as qualitative reporter. The full-length LacZ gene ([http://partsregistry.org/Part:BBa_I732005 I732005]) is too long to be operated in molecular cloning. Therefore, we usually use a short fragment of the full-length LacZ gene called LacZ &alpha;-fragment ([http://partsregistry.org/Part:BBa_I732006 I732006]) as a substitute of the full-length gene(the beta-galactosidase activity can be restored by the rest of LacZ gene in the chromosomal DNA). The beta-galactosidase activity produced by lacZ gene can be observed on X-gal plates by naked eyes, and can also be quantitatively measured using [https://2007.igem.org/USTC/BetaGalactosidaseAssay ONPG Assay].<br />
<br />
== Double Reporter System ==<br />
To combine the advantages of the mentioned two reporter, we construct a double reporter system consisting of both LacZ and fluorescent protein. The following figures show the 3 versions of the system(refer to [http://partsregistry.org/Part:BBa_I732091 BBa_I732091],<br />
[http://partsregistry.org/Part:BBa_I732092 BBa_I732092],<br />
[http://partsregistry.org/Part:BBa_I732093 BBa_I732093]) and version 3, the untagged stable GFP, is our final choice.<br />
<br />
[[Image:ustc_double reporter 1.jpg|thumb|right|192px|left|'''Figure 5''' Version 1 of double reporter system(BBa_I732091).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 2.jpg|thumb|right|192px|left|'''Figure 6''' Version 2 of double reporter system(BBa_I732092).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 3.jpg|thumb|right|192px|left|'''Figure 7''' Version 3 of double reporter system(BBa_I732093).]]<br />
<br style="clear:both;"></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Inputs_and_OutputsUSTC/Inputs and Outputs2007-10-26T07:09:52Z<p>Zhao Yun: </p>
<hr />
<div>= Input Devices =<br />
<br />
PoPS (the flow of RNA Polymerase molecules along DNA) means the current of gene expression. Our logic gates accept PoPS input signals, but we cannot input a PoPS signal directly. We decide to add something into our system as input signal, such as cheminal ligands and light. So a convertor must be built firstly to convert these signals to PoPS signals.<br />
<br />
<br />
<br />
== Candidates of input signals ==<br />
=== Light ===<br />
We decide not to use light signals as the inputs of our system for the following two reasons. <br />
<br />
For one, it would be rather difficult to dynamically control the light intensity in real experiments. Compared with simply adding reagents to the solutions, controlling the input level of light is far more complicated. Still unpleasant is the requirement for the techniques of keeping the light sensors on the cell membrane from frequent exposure. <br />
<br />
For another, since light has seldom been applied as input signals before, there is little relative background knowledge and experience to help us.<br />
<br />
=== IPTG ===<br />
<br />
IPTG is actually the RESET signal of our whole system. Once added, it will let all the repressor-operator pairs in the system no longer function. All the outputs will consequently become 1 as well as all the input repressing signals being automatically ignored. (It just likes the "8888..." on the screen when you reset your calculator or some other digital equipment.)<br />
<br />
=== aTc ===<br />
<br />
ATC is a well studied and widely used inducer of cellular communication. We take a fancy to its stable properties and reasonable price. Certainly, there are still some problems, for example, aTc at a high concentration shows some bacteriostatic activity like Tc(TetraCycline). Unfortunately we have to use concentrated aTc for counteracting the high expression of TetR repressor protein.<br />
<br />
=== AHL ===<br />
<br />
We consider AHL a well-qualified input signal that is normally used in quorum sensing. Actually, we choose 3OC6HSL, a member of AHL family to be the input signal, and its according receptor LuxR to be the sensor. Here we find it no problem to use dilute AHL although LuxR is also high expressing, because LuxR is activator and AHL is its accelerator.<br />
<br />
=== Arabinose ===<br />
<br />
Considering that the promoter of arabinose requires to match and is too much limited by the genotype of the host strain, we finally give up the idea that arabinose would serve as an input signal.<br />
<br />
<br />
----<br />
<br />
== Design of PoPS converters ==<br />
<br />
[[Image:PoPS Converters.jpg|386px|left|'''Figure 1''' Design of PoPS converters.]]<br />
<br style="clear:both;"><br />
<br />
The figure above shows the basic structure of our input device. It functions to convert the input chemical signals to the PoPS (the flow of RNA Polymerase molecules along DNA) signals of the lac repressor A and B, the wires that will actually connect the two main parts of our system together.<br />
<br />
== PoPS-converter parts ==<br />
<br />
[[Image:ustc_atc_pops.jpg|thumb|right|192px|left|'''Figure 2''' The first design of [aTc]->PoPS converter (BBa_I732014).]]<br />
<br style="clear:both;"><br />
<br />
Figure 2 is the first design of our [aTc]->PoPS converter. Fluorescent reporters was added behind. However, we found that the background expression could not be ignored under the fluorescent microscope. It might be due to the LVA tag of Tet repressor which served to help TetR degrade. Therefore, we decide to remove the LVA tag of the Tet repressor like the one shown in Figure 3.<br />
<br />
We take [http://partsregistry.org/Part:BBa_J09855 BBa_J09855] directly from the registry as [AHL]->PoPS converter. Fluorescent expression under different [3OC6HSL] has been tested and the results show that this part is a perfect input device in this study.<br />
<br />
[[Image:USTC_aTc_PoPS_2.jpg|thumb|right|192px|left|'''Figure 3''' The second design of [aTc]->PoPS converter (BBa_I732083).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:USTC_AHL_PoPS.jpg|thumb|right|192px|left|'''Figure 4''' [AHL]->PoPS converter (BBa_J09855).]]<br />
<br style="clear:both;"><br />
<br />
= Output Devices =<br />
For the sake of convenience, we choose LacZ and fluorescent proteins as the corresponding qualitative and quantitative reporters. With LacZ, we can roughly estimate the property of a gate candidate while with fluorescent proteins, we can further examine with a fluorescent microscope candidates that has passed the LacZ test, and finally pick out the best ones.<br />
<br />
== Fluorescent Proteins ==<br />
Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) are used in our system as reporters. In most of the measurements, we use the stable version ([http://partsregistry.org/Part:BBa_E0040 BBa_E0040] and [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]). But these fluorescent proteins are too stable for fast degradation. Therefore, the unstable version with LVA tag have also been synthesized ([http://partsregistry.org/Part:BBa_I732077 BBa_I732077] and [http://partsregistry.org/Part:BBa_I732078 BBa_I732078]) to reduce the half life. However, the untagged fluorescent proteins could better approach our expectation and requirements, so we construct the final version of our reporter system with the stable RFP and GFP.<br />
<br />
== LacZ: Beta-Galactosidase Activity ==<br />
LacZ is widely used as qualitative reporter. The full-length LacZ gene ([http://partsregistry.org/Part:BBa_I732005 I732005]) is too long to be operated in molecular cloning. Therefore, we usually use a short fragment of the full-length LacZ gene called LacZ &alpha;-fragment ([http://partsregistry.org/Part:BBa_I732006 I732006]) as a substitute of the full-length gene(the beta-galactosidase activity can be restored by the rest of LacZ gene in the chromosomal DNA). The beta-galactosidase activity produced by lacZ gene can be observed on X-gal plates by naked eyes, and can also be quantitatively measured [[using]] [https://2007.igem.org/USTC/BetaGalactosidaseAssay ONPG Assay].<br />
<br />
== Double Reporter System ==<br />
To combine the advantages of the mentioned two reporter, we construct a double reporter system consisting of both LacZ and fluorescent protein. The following figures show the 3 versions of the system(refer to [http://partsregistry.org/Part:BBa_I732091 BBa_I732091],<br />
[http://partsregistry.org/Part:BBa_I732092 BBa_I732092],<br />
[http://partsregistry.org/Part:BBa_I732093 BBa_I732093]) and version 3, the untagged stable GFP, is our final choice.<br />
<br />
[[Image:ustc_double reporter 1.jpg|thumb|right|192px|left|'''Figure 5''' Version 1 of double reporter system(BBa_I732091).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 2.jpg|thumb|right|192px|left|'''Figure 6''' Version 2 of double reporter system(BBa_I732092).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 3.jpg|thumb|right|192px|left|'''Figure 7''' Version 3 of double reporter system(BBa_I732093).]]<br />
<br style="clear:both;"></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Inputs_and_OutputsUSTC/Inputs and Outputs2007-10-26T07:09:19Z<p>Zhao Yun: /* Output Devices */</p>
<hr />
<div>= Input Devices =<br />
<br />
PoPS (the flow of RNA Polymerase molecules along DNA) means the current of gene expression. Our logic gates accept PoPS input signals, but we cannot input a PoPS signal directly. We decide to add something into our system as input signal, such as cheminal ligands and light. So a convertor must be built firstly to convert these signals to PoPS signals.<br />
<br />
<br />
<br />
== Candidates of input signals ==<br />
=== Light ===<br />
We decide not to use light signals as the inputs of our system for the following two reasons. <br />
<br />
For one, it would be rather difficult to dynamically control the light intensity in real experiments. Compared with simply adding reagents to the solutions, controlling the input level of light is far more complicated. Still unpleasant is the requirement for the techniques of keeping the light sensors on the cell membrane from frequent exposure. <br />
<br />
For another, since light has seldom been applied as input signals before, there is little relative background knowledge and experience to help us.<br />
<br />
=== IPTG ===<br />
<br />
IPTG is actually the RESET signal of our whole system. Once added, it will let all the repressor-operator pairs in the system no longer function. All the outputs will consequently become 1 as well as all the input repressing signals being automatically ignored. (It just likes the "8888..." on the screen when you reset your calculator or some other digital equipment.)<br />
<br />
=== aTc ===<br />
<br />
ATC is a well studied and widely used inducer of cellular communication. We take a fancy to its stable properties and reasonable price. Certainly, there are still some problems, for example, aTc at a high concentration shows some bacteriostatic activity like Tc(TetraCycline). Unfortunately we have to use concentrated aTc for counteracting the high expression of TetR repressor protein.<br />
<br />
=== AHL ===<br />
<br />
We consider AHL a well-qualified input signal that is normally used in quorum sensing. Actually, we choose 3OC6HSL, a member of AHL family to be the input signal, and its according receptor LuxR to be the sensor. Here we find it no problem to use dilute AHL although LuxR is also high expressing, because LuxR is activator and AHL is its accelerator.<br />
<br />
=== Arabinose ===<br />
<br />
Considering that the promoter of arabinose requires to match and is too much limited by the genotype of the host strain, we finally give up the idea that arabinose would serve as an input signal.<br />
<br />
<br />
----<br />
<br />
== Design of PoPS converters ==<br />
<br />
[[Image:PoPS Converters.jpg|386px|left|'''Figure 1''' Design of PoPS converters.]]<br />
<br style="clear:both;"><br />
<br />
The figure above shows the basic structure of our input device. It functions to convert the input chemical signals to the PoPS (the flow of RNA Polymerase molecules along DNA) signals of the lac repressor A and B, the wires that will actually connect the two main parts of our system together.<br />
<br />
== PoPS-converter parts ==<br />
<br />
[[Image:ustc_atc_pops.jpg|thumb|right|192px|left|'''Figure 2''' The first design of [aTc]->PoPS converter (BBa_I732014).]]<br />
<br style="clear:both;"><br />
<br />
Figure 2 is the first design of our [aTc]->PoPS converter. Fluorescent reporters was added behind. However, we found that the background expression could not be ignored under the fluorescent microscope. It might be due to the LVA tag of Tet repressor which served to help TetR degrade. Therefore, we decide to remove the LVA tag of the Tet repressor like the one shown in Figure 3.<br />
<br />
We take [http://partsregistry.org/Part:BBa_J09855 BBa_J09855] directly from the registry as [AHL]->PoPS converter. Fluorescent expression under different [3OC6HSL] has been tested and the results show that this part is a perfect input device in this study.<br />
<br />
[[Image:USTC_aTc_PoPS_2.jpg|thumb|right|192px|left|'''Figure 3''' The second design of [aTc]->PoPS converter (BBa_I732083).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:USTC_AHL_PoPS.jpg|thumb|right|192px|left|'''Figure 4''' [AHL]->PoPS converter (BBa_J09855).]]<br />
<br style="clear:both;"><br />
<br />
= Output Devices =<br />
For teh sake of convenience, we choose LacZ and fluorescent proteins as the corresponding qualitative and quantitative reporters. With LacZ, we can roughly estimate the property of a gate candidate while with fluorescent proteins, we can further examine with a fluorescent microscope candidates that has passed the LacZ test, and finally pick out the best ones.<br />
<br />
== Fluorescent Proteins ==<br />
Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) are used in our system as reporters. In most of the measurements, we use the stable version ([http://partsregistry.org/Part:BBa_E0040 BBa_E0040] and [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]). But these fluorescent proteins are too stable for fast degradation. Therefore, the unstable version with LVA tag have also been synthesized ([http://partsregistry.org/Part:BBa_I732077 BBa_I732077] and [http://partsregistry.org/Part:BBa_I732078 BBa_I732078]) to reduce the half life. However, the untagged fluorescent proteins could better approach our expectation and requirements, so we construct the final version of our reporter system with the stable RFP and GFP.<br />
<br />
== LacZ: Beta-Galactosidase Activity ==<br />
LacZ is widely used as qualitative reporter. The full-length LacZ gene ([http://partsregistry.org/Part:BBa_I732005 I732005]) is too long to be operated in molecular cloning. Therefore, we usually use a short fragment of the full-length LacZ gene called LacZ &alpha;-fragment ([http://partsregistry.org/Part:BBa_I732006 I732006]) as a substitute of the full-length gene(the beta-galactosidase activity can be restored by the rest of LacZ gene in the chromosomal DNA). The beta-galactosidase activity produced by lacZ gene can be observed on X-gal plates by naked eyes, and can also be quantitatively measured [[using]] [https://2007.igem.org/USTC/BetaGalactosidaseAssay ONPG Assay].<br />
<br />
== Double Reporter System ==<br />
To combine the advantages of the mentioned two reporter, we construct a double reporter system consisting of both LacZ and fluorescent protein. The following figures show the 3 versions of the system(refer to [http://partsregistry.org/Part:BBa_I732091 BBa_I732091],<br />
[http://partsregistry.org/Part:BBa_I732092 BBa_I732092],<br />
[http://partsregistry.org/Part:BBa_I732093 BBa_I732093]) and version 3, the untagged stable GFP, is our final choice.<br />
<br />
[[Image:ustc_double reporter 1.jpg|thumb|right|192px|left|'''Figure 5''' Version 1 of double reporter system(BBa_I732091).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 2.jpg|thumb|right|192px|left|'''Figure 6''' Version 2 of double reporter system(BBa_I732092).]]<br />
<br style="clear:both;"><br />
<br />
[[Image:ustc_double reporter 3.jpg|thumb|right|192px|left|'''Figure 7''' Version 3 of double reporter system(BBa_I732093).]]<br />
<br style="clear:both;"></div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Repressor_Evolution_in_SilicoUSTC/Repressor Evolution in Silico2007-10-26T06:59:08Z<p>Zhao Yun: </p>
<hr />
<div>= Introduction =<br />
<br />
== Protein Design ==<br />
Designing efficient proteins for a broad of different processes is of tremendous practical fun both in science and industry. [[USTC/Repressor_Evolution_in_Silico#References|[1]]]. Computational design is well concerned for its efficiency and convenience. Redesigning the protein-DNA complexes is so important that it is the first step concerned at the beginning of redesigning the active sites. Milestones have been reported[[USTC/Repressor_Evolution_in_Silico#References|[2-5]]]. Here we are trying to construct several artificial repressor-operator pairs to serve as the connecting wires of our system.<br />
<br />
There are several steps in protein design[[USTC/Repressor_Evolution_in_Silico#References|[6]]]. Firstly, generate a random structure with random sequence. Secondly, optimize the structure of the side-chains for each random sequence. Thirdly, give each random sequence a score and select the sequence with the best score. In almost all the conditions, sequence candidates is countless. There will be 20<sup>20</sup> candidates if we redesign 20 positions. Therefore, great efforts have been made to reduce the computation complexity[[USTC/Repressor_Evolution_in_Silico#References|[1,7-14]]], for example, using a rotamer library that is composed of several discrete conformation to represent the status of sidechains, employing pair-wise energy function to score sequences, searching with Monte Carlo, Genetic Algorithm and etc. Another key here is the score function. It functions to give higher scores to sequences performs better in experiments. Up till now, no efficient method in silico has been given to examine the computational design results. One way to examine them is to express the sequences designed in practical experiments.<br />
<br />
== Lac Repressor ==<br />
<br />
[[Image:USTC_1l1m_bio_r_500.jpg|thumb|300px|'''Figure 1''' Solution structure of a dimer of Lac repressor DNA-binding domain complexed to its natural operator O1 ([http://www.rcsb.org/pdb/explore.do?structureId=1L1M From RCSB])]]<br />
<br />
The lac repressor is a DNA-binding protein which inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria[[USTC/Repressor_Evolution_in_Silico#References|[15]]]. There are three distinct regions in the protein. The headpiece, a fragment contains approximately 51 amino acids from its N-terminal together with a helix-turn-helix motif, is the only region that serves to bind DNA. Figure 1 shows the NMR structure of the complex with two headpieces of the lac repressor and its nature operator[[USTC/Repressor_Evolution_in_Silico#References|[16]]]. A series of experiments have shown that the mutation on the 7th, 9th site of the operator can sharply reduce the stability of the complex[[USTC/Repressor_Evolution_in_Silico#References|[15-17]]]. Figure 2 shows the structure of DNA sequence on site 7,8,9 and the residues related to them. From this structure, we come to know that Residue 17 and 18, which are YQ in native repressor sequence, take charge in recognizing the specific DNA sequence. The binding specificity may be adapted by changing the two positions.<br />
<br />
As to the wires exempt from interference, the binding specificity is highly required for the repressor-operator pairs. It is reported that the mutated repressor with VA at Position 17,18 can bind to the transitionally mutated operator on Site 7 [[USTC/Repressor_Evolution_in_Silico#References|[18]]]. Transversion might lead to changes too big for us to rebuild the DNA structure according to the native structure, thus, no mutated lac repressor can bind to it. Therefore, only transition mutation made on the DNA, and the structure was built manually according to native structure and then optimized with gromos96, 43a1 forcefield. In this way, we have obtained four DNA structures. Furthermore, we aims at accomplish in the near future redesigning the recognition region of the repressor for specific bindings to the operator DNA.<br />
<br />
[[Image:USTC_lacI_core_residues.png|thumb|384px|'''Figure 2''' The structure of DNA sequence on site 7,8,9 of the recognition helix of Lac repressor and the residues interaction with them]]<br />
<br />
= Computational Model =<br />
== Score function ==<br />
<br />
Our score function here is a combination of physic-based potentials and knowledge-based potentials. The energy items should be modified to suit the rotamer library. The modified van der waal interaction, hydrogen bond energy, solvent accessible surface area (SASA) and electrostatic interaction are applied as items for the score function.<br />
<br />
=== Linearize van der waal interaction ===<br />
The vdw interaction energy[[USTC/Repressor_Evolution_in_Silico#References|[19]]] between atom i and atom j at the distance of r is:<br />
<br />
[[Image:USTC_formula_vdw.png|center]]<br />
<br />
Clashes in redesigned structures more often occur than those in native structures. Obviously, it is because that side chains of the proteins are represented in a discrete form in artificial structures. A tolerate acception of clash is given by linearizing the replusive item , and the parameters are determined by taking tests.<br />
=== Hydrogen bond energy ===<br />
<br />
The distance between the donor and the acceptor given, the hydrogen bond energy is:<br />
<br />
[[Image:USTC_formula_hbond.png|center]]<br />
<br />
When side chain structures are described in the form of rotamers, the distance between the donor and acceptor changes as well. Moreover, the position of hydrogen atom cannot be obtained in a easy way because of its rotation. Here we give a knowledge-based potential depend on the distance of donor and acceptor in the coarse-grained structures.<br />
<br />
=== SASA and Electrostatic Interaction ===<br />
<br />
The energy items for SASA and electostatic interaction are defined by TM Handel and her co-workers[[USTC/Repressor_Evolution_in_Silico#References|[8]]].<br />
<br />
== Parameterization ==<br />
<br />
We work with different score functions to respectively optimize the side chains of the proteins and to select sequences. It is because that they are totally different in nature. Optimization of side-chains tells the three-dimensional structure of the complex after the amino acids there are changed, and the sequence selection of amino acids shows the more suitable sequence for binding. The role of the two energy items in the two processes are not the same. The parameters for the optimized side chain are obtained by training with 49 structures picked from PDBbind library [[USTC/Repressor_Evolution_in_Silico#References|[20]]], and those for sequence selection are gained by training with the results of directed evolution.<br />
<br />
= Results =<br />
<br />
In this particular problem, only 2 positions in the protein were to be changed. Therefore, there are only 400 candidates. The computational complexity is no longer a problem here. We have optimized the side chains of each candidate, and have worked out the each energy item. With the help of directed evolution, we have found the suitable parameters for sequence selection. Additionally, we predict that some other chains may also work in this situation. Testing experiments have already been in process at the same time.<br />
<br />
= References =<br />
<br />
# Zanghellini, A.; Jiang, L.; Wollacott, A. M.; Cheng, G.; Meiler, J.; Althoff, E. A.; Röthlisberger, D. & Baker, D. (2006), 'New algorithms and an in silico benchmark for computational enzyme design.', <i>Protein Sci</i> 15(12), 2785--2794.<br />
# Dahiyat, B. I. & Mayo, S. L. (1997), 'De novo protein design: fully automated sequence selection.', <i>Science</i> 278(5335), 82--87.<br />
# Kuhlman, B.; Dantas, G.; Ireton, G. C.; Varani, G.; Stoddard, B. L. & Baker, D. (2003), 'Design of a novel globular protein fold with atomic-level accuracy.', <i>Science</i> 302(5649), 1364--1368.<br />
# Looger, L. L.; Dwyer, M. A.; Smith, J. J. & Hellinga, H. W. (2003), 'Computational design of receptor and sensor proteins with novel functions.', <i>Nature</i> 423(6936), 185--190.<br />
# Ashworth, J.; Havranek, J. J.; Duarte, C. M.; Sussman, D.; Monnat, R. J.; Stoddard, B. L. & Baker, D. (2006), 'Computational redesign of endonuclease DNA binding and cleavage specificity.', <i>Nature</i> 441(7093), 656--659.<br />
# Lippow, S. M. & Tidor, B. (2007), 'Progress in computational protein design.', <i>Curr Opin Biotechnol</i> 18(4), 305--311.<br />
# Georgiev, I.; Lilien, R. H. & Donald, B. R. (2006), 'Improved Pruning algorithms and Divide-and-Conquer strategies for Dead-End Elimination, with application to protein design', <i>Bioinformatics</i> 22, e174-83.<br />
# Pokala, N. & Handel, T. M. (2004), 'Energy functions for protein design I: efficient and accurate continuum electrostatics and solvation.', <i>Protein Sci 13(4)</i>, 925--936.<br />
# Gordon, D. B.; Hom, G. K.; Mayo, S. L. & Pierce, N. A. (2003), 'Exact rotamer optimization for protein design', <i>Journal of Computational Chemistry</i> 24, 232-43.<br />
# Holm, L. & Sander, C. (1992), 'Fast and simple Monte Carlo algorithm for side chain optimization in proteins: application to model building by homology', <i>Proteins</i> 14, 213-23.<br />
# Lilien, R. H.; Stevens, B. W.; Anderson, A. C. & Donald, B. R. (2005), 'A novel ensemble-based scoring and search algorithm for protein redesign and its application to modify the substrate specificity of the gramicidin synthetase a phenylalanine adenylation enzyme.', <i>J Comput Biol</i> 12(6), 740--761.<br />
# Pokala, N. & Handel, T. M. (2005), 'Energy functions for protein design: adjustment with protein-protein complex affinities, models for the unfolded state, and negative design of solubility and specificity.', <i>J Mol Biol</i> 347(1), 203--227.<br />
# Shah, P. S.; Hom, G. K. & Mayo, S. L. (2004), 'Preprocessing of rotamers for protein design calculations', <i>Journal of Computational Chemistry</i> 25, 1797-800.<br />
# Street, A. G. & Mayo, S. L. (1998), 'Pairwise calculation of protein solvent-accessible surface areas.', <i>Fold Des</i> 3(4), 253--258.<br />
# Lewis, M.; Chang, G.; Horton, N. C.; Kercher, M. A.; Pace, H. C.; Schumacher, M. A.; Brennan, R. G. & Lu, P. (1996), 'Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.', <i>Science</i> 271(5253), 1247--1254.<br />
# Lehming, N.; Sartorius, J.; Niemöller, M.; Genenger, G.; v Wilcken-Bergmann, B. & Müller-Hill, B. (1987), 'The interaction of the recognition helix of lac repressor with lac operator.', <i>EMBO J</i> 6(10), 3145--3153.<br />
# Sartorius, J.; Lehming, N.; Kisters, B.; von Wilcken-Bergmann, B. & Müller-Hill, B. (1989), 'lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges.', <i>EMBO J</i> 8(4), 1265--1270.<br />
# Salinas, R. K.; Folkers, G. E.; Bonvin, A. M. J. J.; Das, D.; Boelens, R. & Kaptein, R. (2005), 'Altered specificity in DNA binding by the lac repressor: a mutant lac headpiece that mimics the gal repressor.', <i>Chembiochem</i> 6(9), 1628--1637.<br />
# Grigoryan, G.; Ochoa, A. & Keating, A. E. (2007), 'Computing van der Waals energies in the context of the rotamer approximation.', <i>Proteins</i> 68(4), 863--878.<br />
# Wang, R.; Fang, X.; Lu, Y. & Wang, S. (2004), 'The PDBbind database: collection of binding affinities for protein-ligand complexes with known three-dimensional structures.', <i>J Med Chem</i> 47(12), 2977--2980.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Repressor_Evolution_in_SilicoUSTC/Repressor Evolution in Silico2007-10-26T06:38:29Z<p>Zhao Yun: /* Protein Design */</p>
<hr />
<div>= Introduction =<br />
<br />
== Protein Design ==<br />
Designing efficient proteins for a broad of different processes is of tremendous practical fun both in science and industry. [[USTC/Repressor_Evolution_in_Silico#References|[1]]]. Computational design is well concerned for its efficiency and convenience. Redesigning the protein-DNA complexes is so important that it is the first step concerned at the beginning of redesigning the active sites. Milestones have been reported[[USTC/Repressor_Evolution_in_Silico#References|[2-5]]]. Here we are trying to construct several artificial repressor-operator pairs to serve as the connecting wires of our system.<br />
<br />
There are several steps in protein design[[USTC/Repressor_Evolution_in_Silico#References|[6]]]. Firstly, generate a random structure with random sequence. Secondly, optimize the structure of the side-chains for each random sequence. Thirdly, give each random sequence a score and select the sequence with the best score. In almost all the conditions, sequence candidates is countless. There will be 20<sup>20</sup> candidates if we redesign 20 positions. Therefore, great efforts have been made to reduce the computation complexity[[USTC/Repressor_Evolution_in_Silico#References|[1,7-14]]], for example, using a rotamer library that is composed of several discrete conformation to represent the status of sidechains, employing pair-wise energy function to score sequences, searching with Monte Carlo, Genetic Algorithm and etc. Another key here is the score function. Up till now, no efficient method in silico has been given to examine the computational design results. One way to examine them is to express the sequences designed in practical experiments.<br />
<br />
== Lac Repressor ==<br />
<br />
[[Image:USTC_1l1m_bio_r_500.jpg|thumb|300px|'''Figure 1''' Solution structure of a dimer of Lac repressor DNA-binding domain complexed to its natural operator O1 ([http://www.rcsb.org/pdb/explore.do?structureId=1L1M From RCSB])]]<br />
<br />
The lac repressor is a DNA-binding protein which inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria[[USTC/Repressor_Evolution_in_Silico#References|[15]]]. There are three distinct regions in the protein. The headpiece, a fragment contains approximately 51 amino acids from its N-terminal together with a helix-turn-helix motif, is the only region that serves to bind DNA. Figure 1 shows the NMR structure of the complex with two headpieces of the lac repressor and its nature operator[[USTC/Repressor_Evolution_in_Silico#References|[16]]]. A series of experiments have shown that the mutation on the 7th, 9th site of the operator can sharply reduce the stability of the complex[[USTC/Repressor_Evolution_in_Silico#References|[15-17]]]. Figure 2 shows the structure of DNA sequence on site 7,8,9 and the residues related to them. From this structure, we come to know that Residue 17 and 18, which are YQ in native repressor sequence, take charge in recognizing the specific DNA sequence. The binding specificity may be adapted by changing the two positions.<br />
<br />
As to the wires exempt from interference, the binding specificity is highly required for the repressor-operator pairs. It is reported that the mutated repressor with VA at Position 17,18 can bind to the transitionally mutated operator on Site 7 [[USTC/Repressor_Evolution_in_Silico#References|[18]]]. Transversion might lead to changes too big for us to rebuild the DNA structure according to the native structure, thus, no mutated lac repressor can bind to it. Therefore, only transition mutation made on the DNA, and the structure was built manually according to native structure and then optimized with gromos96, 43a1 forcefield. In this way, we have obtained four DNA structures. Furthermore, we aims at accomplish in the near future redesigning the recognition region of the repressor for specific bindings to the operator DNA.<br />
<br />
[[Image:USTC_lacI_core_residues.png|thumb|384px|'''Figure 2''' The structure of DNA sequence on site 7,8,9 of the recognition helix of Lac repressor and the residues interaction with them]]<br />
<br />
= Computational Model =<br />
== Score function ==<br />
<br />
Our score function here is a combination of physic-based potentials and knowledge-based potentials. The energy items should be modified to suit the rotamer library. The modified van der waal interaction, hydrogen bond energy, solvent accessible surface area (SASA) and electrostatic interaction are applied as items for the score function.<br />
<br />
=== Linearize van der waal interaction ===<br />
The vdw interaction energy[[USTC/Repressor_Evolution_in_Silico#References|[19]]] between atom i and atom j at the distance of r is:<br />
<br />
[[Image:USTC_formula_vdw.png|center]]<br />
<br />
Clashes in redesigned structures more often occur than those in native structures. Obviously, it is because that side chains of the proteins are represented in a discrete form in artificial structures. A tolerate acception of clash is given by linearizing the replusive item , and the parameters are determined by taking tests.<br />
=== Hydrogen bond energy ===<br />
<br />
The distance between the donor and the acceptor given, the hydrogen bond energy is:<br />
<br />
[[Image:USTC_formula_hbond.png|center]]<br />
<br />
Describing side chain structure with rotamers is changing the distance between the donor and acceptor too, and the position of hydrogen atom can not be obtained in a easy way because of its rotation. Here we build a knowledge-based potential depend on the distance of donor and acceptor in the coarse-grained structures.<br />
<br />
=== SASA and Electrostatic Interaction ===<br />
<br />
The energy items for SASA and electostatic interaction are defined by TM Handel and her co-workers[[USTC/Repressor_Evolution_in_Silico#References|[8]]].<br />
<br />
== Parameterization ==<br />
<br />
We work with different score functions to respectively optimize the side chains of the proteins and to select sequences. It is because that they are totally different in nature. Optimization of side-chains tells the three-dimensional structure of the complex after the amino acids there are changed, and the sequence selection of amino acids shows the more suitable sequence for binding. The role of the two energy items in the two processes are not the same. The parameters for the optimized side chain are obtained by training with 49 structures picked from PDBbind library [[USTC/Repressor_Evolution_in_Silico#References|[20]]], and those for sequence selection are gained by training with the results of directed evolution.<br />
<br />
= Results =<br />
<br />
In this particular problem, only 2 positions in the protein were to be changed. Therefore, there are only 400 candidates. The computational complexity is no longer a problem here. We have optimized the side chains of each candidate, and have worked out(?) the each energy items. With the help of directed evolution, we have found the suitable parameters for sequence selection. Additionally, we predict that some other chains may also work in this situation. Testing experiments have already been in process at the same time.<br />
<br />
= References =<br />
<br />
# Zanghellini, A.; Jiang, L.; Wollacott, A. M.; Cheng, G.; Meiler, J.; Althoff, E. A.; Röthlisberger, D. & Baker, D. (2006), 'New algorithms and an in silico benchmark for computational enzyme design.', <i>Protein Sci</i> 15(12), 2785--2794.<br />
# Dahiyat, B. I. & Mayo, S. L. (1997), 'De novo protein design: fully automated sequence selection.', <i>Science</i> 278(5335), 82--87.<br />
# Kuhlman, B.; Dantas, G.; Ireton, G. C.; Varani, G.; Stoddard, B. L. & Baker, D. (2003), 'Design of a novel globular protein fold with atomic-level accuracy.', <i>Science</i> 302(5649), 1364--1368.<br />
# Looger, L. L.; Dwyer, M. A.; Smith, J. J. & Hellinga, H. W. (2003), 'Computational design of receptor and sensor proteins with novel functions.', <i>Nature</i> 423(6936), 185--190.<br />
# Ashworth, J.; Havranek, J. J.; Duarte, C. M.; Sussman, D.; Monnat, R. J.; Stoddard, B. L. & Baker, D. (2006), 'Computational redesign of endonuclease DNA binding and cleavage specificity.', <i>Nature</i> 441(7093), 656--659.<br />
# Lippow, S. M. & Tidor, B. (2007), 'Progress in computational protein design.', <i>Curr Opin Biotechnol</i> 18(4), 305--311.<br />
# Georgiev, I.; Lilien, R. H. & Donald, B. R. (2006), 'Improved Pruning algorithms and Divide-and-Conquer strategies for Dead-End Elimination, with application to protein design', <i>Bioinformatics</i> 22, e174-83.<br />
# Pokala, N. & Handel, T. M. (2004), 'Energy functions for protein design I: efficient and accurate continuum electrostatics and solvation.', <i>Protein Sci 13(4)</i>, 925--936.<br />
# Gordon, D. B.; Hom, G. K.; Mayo, S. L. & Pierce, N. A. (2003), 'Exact rotamer optimization for protein design', <i>Journal of Computational Chemistry</i> 24, 232-43.<br />
# Holm, L. & Sander, C. (1992), 'Fast and simple Monte Carlo algorithm for side chain optimization in proteins: application to model building by homology', <i>Proteins</i> 14, 213-23.<br />
# Lilien, R. H.; Stevens, B. W.; Anderson, A. C. & Donald, B. R. (2005), 'A novel ensemble-based scoring and search algorithm for protein redesign and its application to modify the substrate specificity of the gramicidin synthetase a phenylalanine adenylation enzyme.', <i>J Comput Biol</i> 12(6), 740--761.<br />
# Pokala, N. & Handel, T. M. (2005), 'Energy functions for protein design: adjustment with protein-protein complex affinities, models for the unfolded state, and negative design of solubility and specificity.', <i>J Mol Biol</i> 347(1), 203--227.<br />
# Shah, P. S.; Hom, G. K. & Mayo, S. L. (2004), 'Preprocessing of rotamers for protein design calculations', <i>Journal of Computational Chemistry</i> 25, 1797-800.<br />
# Street, A. G. & Mayo, S. L. (1998), 'Pairwise calculation of protein solvent-accessible surface areas.', <i>Fold Des</i> 3(4), 253--258.<br />
# Lewis, M.; Chang, G.; Horton, N. C.; Kercher, M. A.; Pace, H. C.; Schumacher, M. A.; Brennan, R. G. & Lu, P. (1996), 'Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.', <i>Science</i> 271(5253), 1247--1254.<br />
# Lehming, N.; Sartorius, J.; Niemöller, M.; Genenger, G.; v Wilcken-Bergmann, B. & Müller-Hill, B. (1987), 'The interaction of the recognition helix of lac repressor with lac operator.', <i>EMBO J</i> 6(10), 3145--3153.<br />
# Sartorius, J.; Lehming, N.; Kisters, B.; von Wilcken-Bergmann, B. & Müller-Hill, B. (1989), 'lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges.', <i>EMBO J</i> 8(4), 1265--1270.<br />
# Salinas, R. K.; Folkers, G. E.; Bonvin, A. M. J. J.; Das, D.; Boelens, R. & Kaptein, R. (2005), 'Altered specificity in DNA binding by the lac repressor: a mutant lac headpiece that mimics the gal repressor.', <i>Chembiochem</i> 6(9), 1628--1637.<br />
# Grigoryan, G.; Ochoa, A. & Keating, A. E. (2007), 'Computing van der Waals energies in the context of the rotamer approximation.', <i>Proteins</i> 68(4), 863--878.<br />
# Wang, R.; Fang, X.; Lu, Y. & Wang, S. (2004), 'The PDBbind database: collection of binding affinities for protein-ligand complexes with known three-dimensional structures.', <i>J Med Chem</i> 47(12), 2977--2980.</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T06:30:09Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, we assessed its performance by giving it a score:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T05:41:20Z<p>Zhao Yun: /* Repression Model */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Note that <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the difference of binding energy of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
As to a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, its performance can be expressed simply as NOT score in a non-dimension form:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T05:29:53Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Where <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the binding energy difference of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
As to a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximately equal high or low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, its performance can be expressed simply as NOT score in a non-dimension form:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T05:23:31Z<p>Zhao Yun: /* Repression Model */</p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
As to a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Where <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the binding energy difference of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximate equal high and low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, its performance can be expressed simply as NOT score in a non-dimension form:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T05:21:36Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models has been measured, and practical artificial logic promoters are hard to make because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Where <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the binding energy difference of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximate equal high and low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, its performance can be expressed simply as NOT score in a non-dimension form:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
In the situation with a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximately equal high or low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out well-performing bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and loaded into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid constitutively expressing a specific repressor is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strains got in the first step and then selected through double resistance.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are loaded into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator positions. At present only the dual-repressed pattern works well as NOR gate, but it brings us a limitation in wires selecting when assembled into the whole system. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only to put an operator of reverse symmetric structure at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/Logic-Gate_PromotersUSTC/Logic-Gate Promoters2007-10-26T05:00:19Z<p>Zhao Yun: </p>
<hr />
<div>== Cis-acting Bio-Logic Gates ==<br />
<br />
In natural cells, combinational logic computation can be carried out by cis-acting elements [[USTC/Logic-Gate_Promoters#References|[4]]]. Theoretically, dual repressors interacting on two adjacent operators can generate complex logic function as NAND, NOT and NOT [[USTC/Logic-Gate_Promoters#References|[1,2,3]]]. However, seldom of the parameters of these models have been measured, and practical artificial logic promoters are hard to made because of the lack of appropriate inputs. In this project, we simplify these models to reduce the number of parameters, use artificial high-specific repressors based-on Lac repressor [[USTC/Logic-Gate_Promoters#References|[8]]] to serve as inputs, predict possible patterns of logic promoters, construct and test them experimentally, all to attempt to find a systematical way to construct cis-acting bio-logic promoters. As a result, a piece of DNA about 60 – 200bp is able to be built up and to act as a logic gate.<br />
<br />
=== Advatanges of Cis-acting Bio-Logic Gates ===<br />
# Work in vivo and can be genetically inherited<br />
# Can be systematically built up according to several patterns<br />
# Small in scale<br />
#* About 2.0nm in width, 20 - 70nm in length, similar to transistors in present VSLI in size [[USTC/Logic-Gate_Promoters#References|[12]]], sometimes even smaller<br />
# Can be cascaded to implement any complex combinational logic computation<br />
#* And is also able to form sequential circuit<br />
<br />
== Repression Model ==<br />
<br />
[[Image:USTC_RepressionModel.png|thumb|right|300px|'''Figure 1''' (a) Sketch map of solo-repression. (b) Sketch map of co-repression.]]<br />
<br />
Lacramioara Bintu et al. have reported a simple thermodynamic model which can quantify promoter activity under one or more regulatory factors [[USTC/Logic-Gate_Promoters#References|[1,2]]]. In this project, we focus on the multiple changes of promoter activity under the existence of one or two repressors. For a weak promoter, the multiple of its change can be approximately described as a function of different repressor concentrations, inter-operator distances, repressor–operator affinity and repressor-repressor interactions.<br />
<br />
For a promoter containing a single operator site shown in Figure 1(a), the promoter activity under <i>R</i> repressor molecules <i>A(R)</i> is:<br />
<br />
[[Image:USTC_RepressionModel_FC_Solo.png|center]]<br />
<br />
Where <i>A(0)</i> is the promoter activity without repression; <i>&rho;(P)</i> is the solo-repression coefficient of the operator at the position <i>P</i>; <i>&Delta;&epsilon;(O)</i> is the binding energy difference of operator <i>O</i> on specific sites to non-specific sites; <i>N<sub>NS</sub></i> is the number of non-specific sites; and <i>K<sub>B</sub></i> means the Boltzmann constant, <i>T</i> is the temperature.<br />
<br />
For a promoter containing two different operators, of which the relative repressors may be able to interact with each other shown in Figure 1(b), the promoter activity under combinations of two repressors, R<sub>A</sub> and R<sub>B</sub>, is given as:<br />
<br />
[[Image:USTC_RepressionModel_FC_Co.png|center]]<br />
<br />
Where <i>&omega;(P<sub>A</sub>, P<sub>B</sub>)</i> is the co-repression coefficient when O<sub>A</sub> is located at <i>P<sub>A</sub></i>, and O<sub>B</sub> at <i>P<sub>B</sub></i>.<br />
<br />
Concerning a NOT gate which works under approximate equal high and low repressor concentration, R<sub>low</sub>=0 and R<sub>high</sub>=R<sub>H</sub>, its performance can be expressed simply as NOT score in a non-dimension form:<br />
<br />
[[Image:USTC_RepressionModel_NOT_Score.png|center]]<br />
<br />
In the same way, NAND score and NOR score are:<br />
<br />
[[Image:USTC_RepressionModel_NAND_Score.png|center]]<br />
<br />
[[Image:USTC_RepressionModel_NOR_Score.png|center]]<br />
<br />
For a fixed combination of two repressors, R<sub>A</sub> and R<sub>B</sub>, and approximate equal high and low repressor concentration, the logic performance of a promoter is a function of inter-operator distances, repressor–operator affinity and repressor-repressor interactions. By adjusting these parameters, it is possible to find out high-performance bio-logic promoters.<br />
<br />
== Schemes of Bio-Logic Promoters ==<br />
<br />
Dozens of potential bio-logic patterns were experimentally synthesized and tested in solo-repression or co-repression test-bench. Some representative ones are shown and commented as following.<br />
<br />
{| border="1"<br />
|-<br />
|align="center"| '''Scheme'''<br />
|align="center"| '''Test-environment'''<br />
|align="center"| '''Results'''<br />
|align="center"| '''Comments'''<br />
|-<br />
| [[Image:USTC_NANDv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI_LRLa.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data.png|93px]]<br />
| [[USTC/Interference between LacI and LRLa|Interference between inputs]]<br />
|-<br />
| [[Image:USTC_NOTv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_LacI.png|64px]]<br />
|align="center"| [[Image:USTC_NOTv1_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NANDv2a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv2a_Data.png|93px]]<br />
| <font color="orange">Works</font><BR>[[USTC/OperatorPosition|But with slight downstream repression]]<br />
|-<br />
| [[Image:USTC_NANDv2b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[USTC/FailureOfNANDv2b|Failed in<BR>X-gal Assay]]<br />
| [[USTC/OperatorComposition|"Ox7" kind of operators are too weak]]<br />
|-<br />
| [[Image:USTC_NORv1.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv1_Data2.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv3a.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3a_Data.png|93px]]<br />
| [[USTC/InterOperatorDistance|Co-repression is too weak]]<BR>[[USTC/OperatorPosition|Downstream solo-repression is to strong]]<br />
|-<br />
| [[Image:USTC_NANDv3b.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv3b_Data.png|93px]]<br />
| <font color="red">Works</font><br />
|-<br />
| [[Image:USTC_NORv2.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NORv2_Data.png|93px]]<br />
| [[USTC/OperatorPosition|Inefficacy of upstream operator]]<br />
|-<br />
| [[Image:USTC_NANDv4.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_ARL2A0203.png|92px]]<br />
|align="center"| [[Image:USTC_NANDv4_Data.png|93px]]<br />
| [[USTC/HybridOperator|Hybrid operator do not work as expected]]<br />
|-<br />
| [[Image:USTC_NORv3.png|380px]]<br />
|align="center"| [[Image:USTC_RM_ARL4A0604_LacI.png|92px]]<br />
|align="center"| [[Image:USTC_NORv3_Data.png|93px]]<br />
| <font color="red">Works</font><BR>[[USTC/CoRepressedOperator|With a request of co-operator]]<br />
|-<br />
|}<br />
<br />
=== Experiences for Bio-Logic Promoters ===<br />
'''Composition of Operator'''<br />
[[USTC/OperatorComposition]]<br />
<br />
'''Position of Operator'''<br />
[[USTC/OperatorPosition]]<br />
<br />
'''Inter-operator Distance'''<br />
[[USTC/InterOperatorDistance]]<br />
<br />
'''Hybrid Operator'''<br />
[[USTC/HybridOperator]]<br />
<br />
'''Co-repressed Operator'''<br />
[[USTC/CoRepressedOperator]]<br />
<br />
== Repression Assay ==<br />
=== Build Up Promoter Family ===<br />
<br />
[[Image:USTC_PCRBuilding.png|thumb|400px|right|'''Figure ''' PCR Building]]<br />
<br />
Firstly, we extend both sides of the conservative region for transcriptional initiation [[USTC/Logic-Gate_Promoters#References|[9]]] of PlacUV5 [[USTC/Logic-Gate_Promoters#References|[7]]], including -35 box,-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:<br />
# They will never include the restriction enzyme cutting sites that will be involved in the whole study;<br />
# They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;<br />
# They will never present in complicated structures.<br />
<br />
Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.<br />
<br />
Then the promoter fragments are digested with XbaI and BamHI and cloned into repression-reporter plasmid, which contains <i>lacZ</i> alpha fragment and <i>gfp</i> under the promoter insertion site.<br />
<br />
=== Solo-Repression Assay ===<br />
<br />
[[Image:SoloRepressionAssay.png|thumb|right|400px|'''Figure''' Solo-Repression]]<br />
<br />
Two plasmids are used in solo-repression assay. First, a plasmid expressing a specific repressor constitutively is transformed into Top10. Then the promoters to be tested, which contain variant operator compositions and positions, are transformed into the strain got into the first step and selected by double resistances.<br />
<br />
<BR clear="both"><br />
<br />
=== Co-Repression Assay ===<br />
<br />
Promoters to be tested are cloned into double-reporter plasmid and then transformed into the four test strains (CR00, CR01, CR10, CR11). By reading the color of the colonies on plates with X-Gal, and by testing the fluorescence intensity under a fluorescence microscope, we can get the solo-repression and co-repression effects of the two repressors on specific promoters. <br />
[[Image:USTC_CoRepressionAssay.png|thumb|300px|'''Figure''' Co-Repression Assay]]<br />
<br />
{| border="1"<br />
|-<br />
|align="center"|'''Genotype'''<br />
|align="center"|'''Character'''<br />
|align="center"|'''Name'''<br />
|-<br />
|Top10/pT-TERM<br />
|So not express any repressors<br />
|align="center"|CR00<br />
|-<br />
|Top10/pT-ARL4A0604<br />
|Constitutively express ARL4A0604<br />
|align="center"|CR01<br />
|-<br />
|Top10/pT-ARL2A0203<br />
|Constitutively express ARL2A0203<br />
|align="center"|CR10<br />
|-<br />
|Top10/pTet-ARL4A0604-ARL2A203<br />
|Constitutively express ARL4A0604 and ARL2A0203<br />
|align="center"|CR11<br />
|}<br />
<br />
<BR clear="both"><br />
<br />
== Final Results ==<br />
<br />
{|<br />
| [[Image:USTC_BestNAND.png|thumb|200px|Best NAND]]<br />
| [[Image:USTC_BestNOR.png|thumb|200px|Best NOR]]<br />
| [[Image:USTC_BestNOT.png|thumb|200px|Best NOT]]<br />
|}<br />
<br />
=== Suggested Patterns ===<br />
[[Image:USTC_BestSchemes.png|thumb|right|300px|'''Figure 5''' Suggested patterns for NOT, NAND and NOR gates.]]<br />
<br />
'''NAND'''<BR><br />
A NAND Gate requires that two solo-repressions should be weak, and co-repression should be strong. We choose +83.5 to put the upstream operator, to avoid the uncertain activator regions. Another weak operator is put down at the +66.5 site. The relative distance between the two operators is 150, indicating a strong co-repression.<br />
<br />
'''NOR'''<BR><br />
We expected to find a NOR gate with two different operators around the conservative region of a promoter. But there is no available repressor binding site in the upstream of the conservative region based on the observed effect of operator position. At present only the co-repressed pattern works well as NOR gate, but it brings in a limitation in integration. <br />
<br />
'''NOT'''<BR><br />
NOT gate is quite simple, only one symmetric operator at the +10.5 site.<br />
<br />
<BR clear="both"><br />
<br />
== References ==<br />
<br />
1. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J.; Kuhlman, T. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: applications.', <i>Curr Opin Genet Dev</i> 15(2), 125--135.<br />
<br />
2. Bintu, L.; Buchler, N. E.; Garcia, H. G.; Gerland, U.; Hwa, T.; Kondev, J. & Phillips, R. (2005), 'Transcriptional regulation by the numbers: models.', <i>Curr Opin Genet Dev</i> 15(2), 116--124.<br />
<br />
3. Buchler, N. E.; Gerland, U. & Hwa, T. (2003), 'On schemes of combinatorial transcription logic.', <i>PNAS</i> 100(9), 5136--5141.<br />
<br />
4. Davidson, E. H.; Rast, J. P.; Oliveri, P.; Ransick, A.; Calestani, C.; Yuh, C.; Minokawa, T.; Amore, G.; Hinman, V.; Arenas-Mena, C.; Otim, O.; Brown, C. T.; Livi, C. B.; Lee, P. Y.; Revilla, R.; Rust, A. G.; jun Pan, Z.; Schilstra, M. J.; Clarke, P. J. C.; Arnone, M. I.; Rowen, L.; Cameron, R. A.; McClay, D. R.; Hood, L. & Bolouri, H. (2002), A genomic regulatory network for development., <i>Science</i> 295(5560), 1669--1678.<br />
<br />
5. Elledge, S. J. & Davis, R. W. (1989), 'Position and density effects on repression by stationary and mobile DNA-binding proteins.', <i>Genes Dev</i> 3(2), 185--197.<br />
<br />
6. Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R. & Kaptein, R. (2002), 'Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.', <i>EMBO J</i> 21(12), 2866--2876.<br />
<br />
7. Lanzer, M. & Bujard, H. (1988), 'Promoters largely determine the efficiency of repressor action.', <i>PNAS</i> 85(23), 8973--8977.<br />
<br />
8. Lewis, M. (2005), 'The lac repressor.', <i>C R Biol</i> 328(6), 521--548.<br />
<br />
9. Rojo, F. (1999), 'Repression of transcription initiation in bacteria.', <i>J Bacteriol</i> 181(10), 2987--2991.<br />
<br />
10. Saiz, L. & Vilar, J. M. G. (2006), 'DNA looping: the consequences and its control.', <i>Curr Opin Struct Biol</i> 16(3), 344--350.<br />
<br />
11. Sheridan, S. D.; Opel, M. L. & Hatfield, G. W. (2001), 'Activation and repression of transcription initiation by a distant DNA structural transition.', <i>Mol Microbiol</i> 40(3), 684--690.<br />
<br />
12. [http://cnse.albany.edu/News/index.cfm?step=show_detail&NewsID=424 Semiconductor International: 45 to 32 nm: Another Evolutionary Transition.]</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/IntroductionUSTC/Introduction2007-10-26T04:21:11Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:USTC_Biologic vs Electronic.png|thumb|right|Comparison of electronic transistor with DNA helix.]]<br />
<br />
It is charming for biology scientists to implement a computer in biological organisms. Primarily, logic circuits made with biological materials can work in vitro [1]. More to the point, attempts to carry out unique logic gates in vivo have been reported [2]. Here, our team has been trying to work out a systematic method to realize an extensible logical circuit in vivo.<br />
<br />
As the basic elements of the logic circuits, AND, OR and NOT (or NAND, NOR and NOT) gates should be implemented in the first instance. Transcriptional regulation, which is widely found in biological organism, can be considered as a kind of switch. It serves as the basis of the logic gates in vivo. A negative transcriptional regulation is utilized as a NOT gate. The NOR and NAND gates are realized by installing two operator at the proper distance. All the gates are made on a 60~200-bp DNA fragments in this way.<br />
<br />
To transmit signalS between the gates is more complex in vivo than to do so in vitro. The signal messengers should be specific enough to avoid interference among them. Also, their life expectancy should be long enough to finish their work. Proteins are employed here to be the messengers. To possess enough binding specificity with DNA, proteins are screened with computational protein design and directed evolution. Highly qualified wires are thus obtained.<br />
<br />
Furthermore, by means of assembling all our parts, a demonstration system will be made to drive the integrated system to work in vivo.<br />
<br />
----<br />
<br />
1. Georg Seelig, David Soloveichik, David Yu Zhang, Erik Winfree, 'Enzyme-Free Nucleic Acid Logic Circuits', <i>Science</i> Vol. 314. no. 5805, pp. 1585 - 1588 (2006)<br />
<br />
2. Yohei Yokobayashi, Ron Weiss, and Frances H. Arnold, 'Directed evolution of a genetic circuit', <i>PNAS</i> (2002) vol 99 no 26 16587–16591</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-26T04:16:04Z<p>Zhao Yun: /* Research Work */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work in iGEM==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
In detail, I have worked on four parts.<br />
<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
<br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
<br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to ----<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-26T03:57:18Z<p>Zhao Yun: /* Research Work */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work ==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
In detail, I have worked on four parts.<br />
<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
<br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
<br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
<br />
Finally, I succeed to find the sequence pattern for NAND and NOT promoters, together with a not so perfect NOR promoter, in a systemetic method.<br />
<br />
<br />
More detailed process about promoter design and logic gate construction, please refer to ----<br />
<br />
Without doubt I must mention that all the work listed above is accomplished with much help of my senior fellow apprentices. I am really grateful for their help and kindness.<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-26T03:38:08Z<p>Zhao Yun: /* Research Experience */</p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work ==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
<br />
<br />
== Research Experience ==<br />
<br />
(A)USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
(B)Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
(C)Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yunhttp://2007.igem.org/wiki/index.php/USTC/ZhaoYunUSTC/ZhaoYun2007-10-26T03:37:25Z<p>Zhao Yun: </p>
<hr />
<div>[[Image:ustc_zhaoyun.jpg|200px|thumb]]<br />
<br />
<br />
== Contact: ==<br />
'''Address:'''<br />
<br />
Room 438, Life Science Building, University of Science and Technology of China<br />
<br />
Hefei, Anhui, P.R.China (230027)<br />
<br />
<br />
'''Email:''' <br />
<br />
[mailto:zhaoyun@mail.ustc.edu.cn zhaoyun@mail.ustc.edu.cn] (preference) <br />
<br />
or [mailto:zhaoyunenator@gmail.com zhaoyunenator@gmail.com]<br />
<br />
<br />
'''Tel:'''<br />
<br />
+86-551-3602469<br />
<br />
'''Mobile:'''<br />
<br />
+86-13866776861<br />
<br />
== Research Work ==<br />
<br />
My major work in iGEM is to design and biologically implement three logic promoters: NAND,NOR and NOT. I attempt to systematically build up a procaryotic promoter family whose members contain different operons, that is, operons with different nucleotide sequences and different relative locations. We tested the expression activity under various combined signals of upstream repressors, and systematically study on how different operons influence the expression activity of repressors. <br />
<br />
In detail, I have worked on four parts.<br />
The first one is that I designed to use PCR method to build up the procaryotic promoter family. <br />
The second one is that I measured and compared different repression efficiency according to the different locations of the two operons. <br />
The third is that I measured and compared different repression efficiency according to the different nucleotide sequence of the two operons, and have found a way to alter the combination intensity of repressors.<br />
And the fourth one is that I discussed ranges of several parameters that are suitable for NAND, NOR, NOT Gate, with the help of data of DNA looping reported on previous papers. And I have attempted to synthesize and test the targeted artificial logic promoters.<br />
<br />
<br />
== Research Experience ==<br />
<br />
USTC iGEM Team Member<br />
Project: “Extensible Logic Circuit in Bacteria”. Succeed to find out the patterns for three bio-logic promoters, NAND, NOR, and NOT.<br />
<br />
Undergraduate Research Project<br />
Thesis title: “Artificial Bio-Logic Promoters, Model and Implementation”<br />
<br />
Undergraduate Internship<br />
Lab of Computational Biology, USTC<br />
Supervisor: Prof. Haiyan Liu<br />
NNSFC(National Nature Science Foundation) Projects involved: “Theoretical Design and Experimental Analysis of Artificial Biological Network based on cell-cell communication”<br />
<br />
<br />
== Academic Activities ==<br />
<br />
Presentation in Tianjin iGEM TTT Workshop<br />
Title: “Another Implementation of A Half Adder”</div>Zhao Yun