Peking The Projects

From 2007.igem.org

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(Towards Self-differentiated Bacterial Assembly Line)
 
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{| style="color:#000000; background-color:#e6fff8;" cellpadding="5" cellspacing="1" border="0" bordercolor="#1100ff" width="62%"
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[[Image:Peking_Final_logo.jpg|310px]][[Image:Peking Head banner-3.jpg|570px]]
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{| style="color:#000000; background-color:#EEf8b3;" cellpadding="5" cellspacing="1" border="0" bordercolor="#1100ff" width="933"
![[Peking|Peking]]
![[Peking|Peking]]
![[Peking The Projects|The Projects]]
![[Peking The Projects|The Projects]]
![[Peking The Community|The Team]]
![[Peking The Community|The Team]]
![[Peking The Fun|Misc & Fun]]
![[Peking The Fun|Misc & Fun]]
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![http://openwetware.org/wiki/IGEM:Peking/2007 Peking OWW]
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![http://www.openwetware.org/wiki/IGEM:Peking/2007 Our OWW]
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![[Peking Thanks|Thanks]]
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![[Peking Thanks|Acknowledgement]]
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|}
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=Towards Self-differentiated Bacterial Assembly Line=
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Our projects concern with the ability for bacterial cells to '''differentiate''' out of homogeneous conditions into populations with the '''division of labor'''. We aim at devices conferring host cells with the ability to form cooperating groups spontaneously and to take consecutive steps sequentially even when the genetic background and environmental inputs are identical. To break the mirror in such homogeneous condition, we need two devices respectively responsible for '''temporal''' and '''spatial''' differentiation. The implementation and application of such devices will lead to a picture of bioengineering where complex programs consisted of sequential steps (structure oriented programs) and cooperating agencies (forked instances of a single class, object and event oriented) can be embedded in a single genome.
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==Hop Count with Conjugation==
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<br>
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Hop counting is a cell-cell communication system in which the signals transfered among the cells record the number of times of transfer. we have adapted natural conjugation systems as the communication device, and the conjugative plasmids as the vector for the transfered signal.
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Both the functions and design principles of the two devices are described in the below sections, the functions are presented by two figurative .gif animations to present what they do and how they make cells differentiate in the dimension of space and time. '''Detailed design and Experimental results are presented in two separate pages linked below'''.  
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With the rolling-cycle replication of conjugation, independent tandem oriT (<bbpart>BBa_J01002</bbpart> and more) pairs can be inserted into the same plasmids, losing one tandem region at each single conjugation event.
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Sequential conjugation and deletion of the different oriT pairs can be achieved by fine control of the expression of the cis-acting elements of the independent conjugation systems.Besides intellectual interests, these systems are the core parts of controllable and encoded local cell-cell communication, which can be used in potential pattern formation and material weaving applications.
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==Push-on-push-off Switch==
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<br>
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'''Achievement:'''
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About the name of projects, the two devices are named after their respective functions, but they are common for breaking symmetry, which is interesting and ubiquitous in science. Although this "differentiation" process resemble the development of multicellular organism, we tend to use a more bioengineering style analogy: assembly line. Or maybe after some years from now, this will not be just an analogy.
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[[Image:Peking Pass.JPG|60px]]'''Mathematical Model'''
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[[Image:Peking Pass.JPG|60px]]'''Bistable Switch Construction'''
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[[Image:Peking Pass.JPG|60px]]'''Bistable Switch Test'''
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[[Image:Peking Pass.JPG|60px]]'''Nor-gate Construction'''
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[[Image:Peking Pass.JPG|60px]]'''Nor-gate Test'''
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[[Image:Peking Notdoneyet.jpg|65px]]'''Final Assembling'''
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<table border="0" cellpadding="20" width=95% style="background-color:#f7fbd7;">
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<tr>
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<th><div style="font: normal 1.2em georgia, serif">[[Peking_Hop-Count|'''Hop Count with Conjugation''']]<br></div>
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Click [[Peking_Hop-Count|'''HERE''']] or visit [http://www.openwetware.org/wiki/IGEM:Peking/2007 '''OUR OWW site'''] for all the details of this part!
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</th>
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<th><div style="font: normal 1.2em georgia, serif">[[Peking_Push-on-push-off|'''Push-on-push-off Switch''']]<br></div>
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----
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Click [[Peking_Push-on-push-off| '''HERE''']] or visit [http://www.openwetware.org/wiki/IGEM:Peking/2007 '''OUR OWW site'''] for all the details of this part!
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</th>
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</tr>
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<tr>
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<td align="center">[[Image:Peking_Count_Function.gif]]</td>
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<td align="center">[[Image:Peking_Switch_Function.gif]]</td>
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</tr>
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<tr>
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<td valign="top">
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Hop counting is a cell-cell communication system in which the signals transfered among the cells record the number of times of transfer. With this system, cells can use the transmitted signal as topological cues of their neighborhood, and start the first step of differentiation and cooperation.
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===='''Abstract:''' ====
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We have adapted the natural conjugation systems to a communication device where the conjugative plasmids as the vector for the transfered signal. The numbering signals are directly encoded on the transmitted DNA sequence.
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We are going to engineer the E. coli cell to behave as a '''push-on push-off switch'''. We have designed a gene regulatory circuit that senses Ultra-Violet light (UV) irradiation as input signal. Under intermittent irradiation, two reporter genes are to be induced alternately. The circuit can be divided into two associated parts: '''a bistable switch''' and a '''NOR gate'''. The NOR gate is controlled by the bistable switch and the input signal. On the other hand, the output of the NOR gate can switch the bistable state from one to the other. Thus far, we have constructed a series of NOR gates and one bistable switch.
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Utilizing a special character of the rolling-cycle replication of DNA during conjugation, we use a design where independent tandem oriT (<bbpart>BBa_J01002</bbpart> and more) pairs are inserted into the same plasmids, which lost one tandem region at one conjugation event. Thus the number of conjugation are recorded on the presence/absence of regions on the plasmid of arbitrary length.
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Sequential conjugation and deletion of the different oriT pairs can be achieved by fine control of the expression of the trans-acting factors of the independent conjugation systems. In fact, we inserted the enzymes directly responsible for the initiation of conjugative DNA replication (the relaxase) followed by a terminator between the corresponding oriT. The expression of all the enzymes are under control of a unified promoter. The terminators ensure only the relaxase of the most upstream undeleted tandem oriT regions are expressed. This design guarantees the independent conjugative systems are deleted at a specified order.
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====Gene Circuit Components:====
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Fig.1 shows the scheme of the gene circuit. The '''Bistable Switch''' is composed of CI and CI434 which repress each other in transcription. The '''NOR Gate''' is a promoter repressed by both LacI and LexA. These two parts are connected by LacI and CI(ind-). UV irradiation induces SOS response in E. coli, causing rapid degradation of LexA, CI and CI434. CI(ind-) is a mutation of CI which functions the same as CI but is not readily degraded in SOS response.
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*[[Image:Peking Swich fig.1.JPG|500px]]
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====Mathematical model ====
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A set of ordinary differential equations (ODEs) are used to describe the dynamic behavior of this circuit after UV irradiation. As shown in Fig 2, initially the cell is expressing CI-GFP-LacI. CI434-RFP is repressed by CI, and CI(ind-) is repressed by both LexA and LacI. We call this '''state 1'''. When the cell is exposed to UV irradiation, LexA and CI are degraded. In the absence of CI, LacI transcription attenuates. Meanwhile, LacI concentration decreases gradually owning to the cell growth. At the same time, as the DNA damage produced by UV being repaired, LexA, CI, and CI434 tend to re-accumulate. As a result, during the SOS response and thereafter, CI(ind-) is always repressed, firstly by LacI and then by LexA. At this moment, because the promoter PR is stronger than PRM, CI434 accumulates faster and represses CI expression. The cell turns to '''state 2''', in which CI434-RFP is high and CI-GFP-LacI is low.
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When the cell is exposed to UV irradiation again, LexA is degraded, and in the absence of both LexA and LacI, CI(ind-) is expressed. Before CI(ind-) is turned off by LacI and LexA, it helps to repress CI434, and induce the expression of CI, turning the cell back to '''state 1'''.
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According to the simulation result, the circuit does have the potential to behave like a push-on push-off switch.
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[[Image:Peking Swich fig.2.JPG]]
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====Constructing the Bistable Switch====
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We modify the wild type PRM/PR region of lambda phage so that it can be repressed by CI and CI434 in opposite transcription direction. As shown in Fig 3(a), two different promoter designs are considered. The first is to replace OR3 with two CI434 binding site, while the second is to add them after OR3. Three SD signals are selected as candidates. pSB3K3 plasmid is chosen to carry the switch. The plasmid is transformed into strain DH5alpha. The culture is then plated over night. It turns out that the first promoter design shows bistable characteristic, namely, a fraction of the colons express RFP while others express EGFP (Fig 3 (b) and (c)). 
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====Constructing the NOR Gate====
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We combine the SOS gene promoter with LacI binding site so that it can be repressed by both LacI and LexA. In order to obtaining a satisfying NOR gate, we make 15 combinations to cover various possibilities. As shown in Fig 4, LacO1, LacO2, LacO3 are LacI binding sites of different affinity. recAop and sulAop are LexA binding sites from two SOS genes, sulA and recA respectively. ‘designed’ is the most consensus LexA binding site. Plac, Prec, PsulA are wild type promoters (from -35 region to +1) of lac operon, recA and sulA respectively. EGFP is constructed downstream to these promoters in plasmid pLX007, serving as a reporter.
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[[Image:Peking Swich fig.4.JPG|600px]]
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====Measuring the NOR Gates====
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[[Image:Tandem_conjugation.jpg|400px]]
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The plasmid is transformed into strain CAJ64 ((del)(colB-lacI)3, relA1, spoT1, trpT171(UGAs)). The culture is irradiated in mid-log phase. Thereafter, samples are taken every half hour. The cell length and EGFP level are measured by FACS.
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*SOS response in CAJ64. During SOS response, the length of the cell correlates with LexA protein level. When LexA level is low, a SOS gene sulA is activated and represses cell division, so that the cell grows longer.  From Fig 5, we can infer that LexA level decreases within half an hour and starts to re-accumulate after 2 hours. 
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[[Image:Peking Swich fig.5.JPG|500px]]
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*In order to compare ‘sulAop’, ‘recAop’ and ‘designed’, we measure the response curve in the absence of LacI. As shown in Fig 6, the promoter with two ‘sulAop’ has both large induction fold and high expression level.
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[[Image:Peking Swich fig.6.JPG|500px]]
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*Based on the results above, we choose SD1, SD2, SD3 to have further testing. A plasmid with LacI is transformed into CAJ64. IPTG is added prior to UV irradiation, to vary the effective level of LacI. As shown in Fig 7, SD2 behaves most similar to a NOR Gate.
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[[Image:Peking Swich fig.7.JPG|500px]]
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====Summary ====
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We have constructed and tested the '''Bistable Switch''' and '''NOR Gate''' which are the basic elements of the push-on push-off switch. The mathematical model also sheds light on how to connect these two elements together to make the circuit work. The relative abundance of various regulators can be fine-tuned by changing the SD signals which we have measured. Our next step would be combine the two parts and realize an integrate system working as expected
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Currently, we have tested and constructed three sets of independent conjugative systems and validated the feasibility of our design with the F system. The next step is to do the 3-step conjugation-counting process in a single strain.
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</td>
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<td valign="top">
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Push-on-push-off switches are binary switches flipping to different direction under the same impulses, depending on the previous states it resides. It differed from a toggle switch that the forces flip the switch back and force are exactly the same. This allows the cells to do sequential logic utilizing both the information of present and pst events.
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'''Email us''' at ''igem-pku AT googlegroups DOT com''
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We have designed a gene regulatory circuit that senses Ultra-Violet light (UV) irradiation as input signal. Under intermittent irradiation, two reporter genes are to be induced alternately. The circuit can be divided into two associated parts: '''a bistable switch''' and a '''NOR gate'''. The bistable switch resembles a toggle switch where the NOR gate is controlled by the bistable switch and the input signal. On the other hand, the output of the NOR gate can switch the bistable state from one to the other. In this way, the NOR gate combined the information of present inputs and the previous states of the system, and thus flipping the switch in different directions based on the previous events, even though the present inputs are always the same.
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'''External links'''
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[[Image:Peking Swich fig.1.JPG|400px]]
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[https://2006.igem.org/wiki/index.php/Main_Page iGEM] |
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[https://2007.igem.org/Main_Page iGEM 2007] |
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[[IGEM2007 Team List|Team List]] |
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[http://partsregistry.org Parts] |
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[http://openwetware.org/wiki/IGEM:Peking/2007 Peking OWW] |
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[http://www.pku.edu.cn Peking University]
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<html><a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/Peking" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/Peking" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" />
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Thus far, we have constructed and tested a series of NOR gates and one bistable switch, successfully obtaining the desired dynamics for the previous design(these dynamics may also be very useful to many other applications). The next is to combine the two together and tune the system to obtain the desired function.
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</a></html>
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</td>
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</tr>
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</table>
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</div>

Latest revision as of 02:07, 27 October 2007

Peking Final logo.jpgPeking Head banner-3.jpg

Peking The Projects The Team Misc & Fun [http://www.openwetware.org/wiki/IGEM:Peking/2007 Our OWW] Acknowledgement

Towards Self-differentiated Bacterial Assembly Line

Our projects concern with the ability for bacterial cells to differentiate out of homogeneous conditions into populations with the division of labor. We aim at devices conferring host cells with the ability to form cooperating groups spontaneously and to take consecutive steps sequentially even when the genetic background and environmental inputs are identical. To break the mirror in such homogeneous condition, we need two devices respectively responsible for temporal and spatial differentiation. The implementation and application of such devices will lead to a picture of bioengineering where complex programs consisted of sequential steps (structure oriented programs) and cooperating agencies (forked instances of a single class, object and event oriented) can be embedded in a single genome.


Both the functions and design principles of the two devices are described in the below sections, the functions are presented by two figurative .gif animations to present what they do and how they make cells differentiate in the dimension of space and time. Detailed design and Experimental results are presented in two separate pages linked below.


About the name of projects, the two devices are named after their respective functions, but they are common for breaking symmetry, which is interesting and ubiquitous in science. Although this "differentiation" process resemble the development of multicellular organism, we tend to use a more bioengineering style analogy: assembly line. Or maybe after some years from now, this will not be just an analogy.


Click HERE or visit [http://www.openwetware.org/wiki/IGEM:Peking/2007 OUR OWW site] for all the details of this part!


Click HERE or visit [http://www.openwetware.org/wiki/IGEM:Peking/2007 OUR OWW site] for all the details of this part!

Peking Count Function.gif Peking Switch Function.gif

Hop counting is a cell-cell communication system in which the signals transfered among the cells record the number of times of transfer. With this system, cells can use the transmitted signal as topological cues of their neighborhood, and start the first step of differentiation and cooperation.

We have adapted the natural conjugation systems to a communication device where the conjugative plasmids as the vector for the transfered signal. The numbering signals are directly encoded on the transmitted DNA sequence. Utilizing a special character of the rolling-cycle replication of DNA during conjugation, we use a design where independent tandem oriT (BBa_J01002 and more) pairs are inserted into the same plasmids, which lost one tandem region at one conjugation event. Thus the number of conjugation are recorded on the presence/absence of regions on the plasmid of arbitrary length. Sequential conjugation and deletion of the different oriT pairs can be achieved by fine control of the expression of the trans-acting factors of the independent conjugation systems. In fact, we inserted the enzymes directly responsible for the initiation of conjugative DNA replication (the relaxase) followed by a terminator between the corresponding oriT. The expression of all the enzymes are under control of a unified promoter. The terminators ensure only the relaxase of the most upstream undeleted tandem oriT regions are expressed. This design guarantees the independent conjugative systems are deleted at a specified order.

Tandem conjugation.jpg

Currently, we have tested and constructed three sets of independent conjugative systems and validated the feasibility of our design with the F system. The next step is to do the 3-step conjugation-counting process in a single strain.

Push-on-push-off switches are binary switches flipping to different direction under the same impulses, depending on the previous states it resides. It differed from a toggle switch that the forces flip the switch back and force are exactly the same. This allows the cells to do sequential logic utilizing both the information of present and pst events.

We have designed a gene regulatory circuit that senses Ultra-Violet light (UV) irradiation as input signal. Under intermittent irradiation, two reporter genes are to be induced alternately. The circuit can be divided into two associated parts: a bistable switch and a NOR gate. The bistable switch resembles a toggle switch where the NOR gate is controlled by the bistable switch and the input signal. On the other hand, the output of the NOR gate can switch the bistable state from one to the other. In this way, the NOR gate combined the information of present inputs and the previous states of the system, and thus flipping the switch in different directions based on the previous events, even though the present inputs are always the same.

Peking Swich fig.1.JPG

Thus far, we have constructed and tested a series of NOR gates and one bistable switch, successfully obtaining the desired dynamics for the previous design(these dynamics may also be very useful to many other applications). The next is to combine the two together and tune the system to obtain the desired function.