Edinburgh/DivisionPopper/Design

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'''MENU''' :[[Edinburgh/DivisionPopper| Introduction]] | [[Edinburgh/DivisionPopper/References|Background]] | [[Edinburgh/DivisionPopper/Applications|Applications]] | [[Edinburgh/DivisionPopper/Design|Design]] | [[Edinburgh/DivisionPopper/Realization|Realization]] | [[Edinburgh/DivisionPopper/Modelling|Modelling]] | [[Edinburgh/DivisionPopper/Status|Status]] | [[Edinburgh/DivisionPopper/Conclusions|Conclusions]]
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'''MENU''' :[[Edinburgh/DivisionPopper| Introduction]] | [[Edinburgh/DivisionPopper/References|Background]] | [[Edinburgh/DivisionPopper/Applications|Applications]] | [[Edinburgh/DivisionPopper/Design|Design&Implementation]] | [[Edinburgh/DivisionPopper/Modelling|Modelling]] | [[Edinburgh/DivisionPopper/Status|Wet Lab]] | [[Edinburgh/DivisionPopper/SBApproach|Synthetic Biology Approach]] | [[Edinburgh/DivisionPopper/Conclusions|Conclusions]]  
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In the Desing phase we define which are the components that compose the device and which are the mechanisms and dynamics we aspect to observe. According to Synthetic Biology guidelines, we decouple the design phase from the realization phase. In particular, we work here at the abstraction level of devices and parts. The parts level is treated only in terms of logical and functional property, thus not going in the detail of actual biological parts. The decision about what biological parts to use and the DNA level details (implementation) is discussed in the [[Edinburgh/DivisionPopper/Realization|Realization]] section.
+
 +
In the Design section we define the architecture (design), the biology elements (implementation) and the mechanisms (dynamics) of the device. According to the [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi abstraction levels hierarchy],  we propose a view of the Division PoPper at the level of device, parts and DNA. Regarding to the dynamics, we explain which biological processes are involved and how they cooperate in defining the behaviour of the device. The logic design and implementation phases have been decoupled.
__TOC__
__TOC__
 +
 +
==Design==
==Design==
-
The Division PoPper functional parts are:
+
In this section we explain the functional components of the device and how they are connected logically and physically. This view of the device follows the [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi abstraction hierarchy] proposed in the Registry of Parts.
-
* two dif sites sequences.
 
-
* a promoter A that can be repressed by protein A.
 
-
* a coding region for protein A.
 
-
* a promoter B that can repressed by protein B.
 
-
* a coding region for protein B.
 
-
* a promoter P.
 
-
[[Image:Division pulser.png|800px]]
+
{| width="20%" align="right" style="text-align:center"
 +
|-
 +
|'''Device abstraction level of the Division Counter'''
 +
|-
 +
|[[Image:Devicelevel.png|350px]] The Division PoPper device generates an input in the form of PoPS signal.
 +
|}
-
This device outputs a PoPS pulse at each cell division.
 
-
It can be hooked up to another device such as a counter.
 
-
For this device to work, dif site-enclosed DNA needs to flip but once per division. Like all recombination sites, dif-sites are directional and bacteria uses directly repeated dif-sites to resolve genome dimers. Research shows that this resolution occurs only at septation, and we use this temporal control to inverse a plasmid-kept sequence to induce different functions.
+
===Device abstraction level ===
-
In its current configuration, <font color="blue">'''Promoter A'''</font> is repressed by continually produced <font color="blue">'''Repressor A'''</font>. At this time, no <font color="orange">'''Repressor B'''</font> is produced.
+
-
As cell division occurs, DNA enclosed by two inverted dif sites is reversed. Initially there is no <font color="orange">'''Repressor B'''</font> present in the cell so <font color="orange">'''Promoter B'''</font> produces PoPS output. As time passes, <font color="orange">'''Repressor B'''</font> production is turned on and 'turns off' <font color="orange">'''Promoter B'''</font>. Meanwhile <font color="blue">'''Repressor A'''</font> is no longer produced and degrades so that, at the next division, <font color="blue">'''Promoter A'''</font> can produce PoPS output and the process repeats.
+
The Division PoPper is a device, because it is a functional element that can be further combined with other devices by the use of a standard  PoPS signal. The device has no formal input but its behaviour is triggered by an "external" stimulus: the cell division.
 +
The output is a PoPS signal that is supposed to be always zero (not considering leaking issues in the promoters) apart from the short period immediately subsequent to a cell division event. In this case the device generates a pulse: the PoPS signal reaches a non-zero level for a period of time, returning to zero afterward. The function of the device is simple: to inform a downstream device that a division has happened by sending a pulse signal. We suggest some system configurations in the [[Edinburgh/DivisionPopper/Applications|Applications]] section.
-
==Assumptions==
 
-
* The Dif sites flip only once during each cell division
 
-
* Repressors A and B are produced and degrade faster than the cell cycle
 
-
* Promoter B does not interfere with production of repressor A
 
-
==Proof of Concept==
 
-
As stated before, the device relies on the dif site flipping only once per cell division. To test whether or not this actually happens, we have devised two simpler experiments.
 
-
===Exp 1===
 
-
[[Image:Divisiontest1.png|300px|float|right]]
 
-
This experiment will prove whether or not the DNA between the two dif sites flip at all during cell division. If the a flip occurs, then the direction the promoter operates will be changed and GFP will be expressed.
 
-
===Exp 2===
 
-
[[Image:Edinburgh_Divisiontest2.png|300px|float|left]]
 
-
This investigates the number of flips that occurs during division and will require rapidly degrading fluorescent proteins.
 
-
After each division we expect to see a change in colour as the promoter activates a different reporter.
 
-
===Exp 3===
 
-
Currently we are planning to test how the dif sites flip using exps 1 & 2. The final device requires a few more parts, each of which needs to be tested.
+
{| width="20%" align="right" style="text-align:center"
 +
|-
 +
|'''Part abstraction level (Design)'''
 +
|-
 +
|[[Image:Partslevel(logic).png|350px]] Design of the part structure in the Division PoPper device.
 +
|}
-
We might wish to test the repressors without the dif sites
+
===Part abstraction level ===
-
[[Image:Edinburgh_Division_pulser.invert.png|500px]]
 
-
The repressor part of the system is simply a pair of inverters.
 
-
There are 3 basic types of inverter in the registry:
+
At the level of part abstraction we define the functional parts of the device: regulatory regions, coding regions, terminators and so on. Instead of proposing directly the actual biological entities, we propose here a logic view. Strictly speaking, we define entities by their attributes and abilities, but without mapping them to real biological objects (his is done in the Implementation section below). The motivation is to separate the functional design from the realization choices. 
-
* TetR          BBa_Q04400 “this inverter functions well. [jb, 5/24/04]”
+
-
* CI (Lambda) BBa_Q04510 “this inverter functions well. [jb, 5/24/04]”
+
-
* LacI BBa_Q04121 "a strong 'on' state with significant background in the 'off' state. [jb,5/24/04]”
+
-
[[Image:Edinburgh Divisiontest3.png|500px|float|right]]
+
'''The Division PoPper functional parts are:'''
-
'''exp 3 design'''
+
* two recombinase site sequences: Rec.
 +
* a promoter A that can be repressed by protein A: promoter A.
 +
* a coding region for protein A: repressor A.
 +
* a promoter B that can be repressed by protein B: promoter B.
 +
* a coding region for protein B: repressor B.
 +
* a promoter P that is constitutively active (expressing).
-
This will test to see if promoter B causes problems with promoter 0's promoting repressor A and tests the standard repressor inverter found in the registry.
 
-
'''Parts required:'''
+
'''The functional ability that these entities should have are:'''
-
* Promoter 0 – suggest BBa_I0500 (arabinose) – Plate 2,  9I
+
* the region between Rec recombinase site sequence is inverted at each cell division.
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* Promoter B – reverse complimentary lacI promoter – Used in exp 1
+
* the protein A and protein B degradation rates should be fast enough to degrade them rapidly when not expressed.
-
* Inverter – suggested BBa_Q04510 (CI (Lambda)) – Plate 2, 13K
+
* the protein A and protein B effects on (respectively) the promoter A and B expression capacity should be strong.
-
* Reporter – use same as experiment 1
+
* promoter P should be strong in its constitutive expression.
-
==Biobricks Required==
 
-
===Biobricks From Registry===
+
'''The physical configuration:'''
-
{|style="width:70%; background-color:#fcaf3e;" cellpadding="10"  
+
The design relies on the alternate expression of the two strands of the DNA segment in between the Recombinase sites. Promoter P is placed outside the "flipping region" and controls its downstream region in the upper strand. The output of the device is given by the PoPS activity on the upper strand downstream from the second recombinase site. Since the region in between the recombinase sites  flips, the output is controlled alternatively by the two DNA strands. In one strand the parts sequence is: repressor A, terminator, Promoter A. In the other the sequence is: repressor B, terminator, Promoter B.
-
! Part || For use in || Brick used
+
 
 +
 
 +
'''The dynamics:'''
 +
 
 +
As cell division occurs, DNA enclosed by two inverted Recombinase sites is inverted. Initially the device is in the configuration with Repressor A and Promoter A on the upper strand. There is no Repressor A present in the cell, so Promoter A produces a constant PoPS output. As time passes, Repressor A accumulates and 'turns off' Promoter B. Meanwhile Repressor B is no produced and degrades so that, at the next division, Promoter B can produce PoPS output and the process repeats. This mechanism can be seen as the sequence of three phases: Forward, Flipping and Backward. We explain for each phases the dynamics:
 +
 
 +
{| border="1" cellpadding="20" cellspacing="0"
 +
!Configuration
 +
!Processes
 +
!Output
|-
|-
-
|Reporter || Exp1, Exp2, Exp3|| [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] - Fast Degrading ECFP
+
|[[Image:PoPperforward.png|350px]]
 +
| During this phase, the output of the device is controlled by Promoter A. We assume there is no Repressor A present initially. Thus Promoter A generates a flux of PoPS output. After a certain amount of time, Repressor A builds up, since the constitutive promoter P placed upstream controls is expression. When the concentration of Repressor A is high enough, the flux of PoPS from the output promoter goes to zero. Meanwhile, the expression of Repressor B is not active. Thus the concentration of Repressor B should decrease to a low level.
 +
| Controlled by Promoter A.
|-
|-
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|Inverter || Exp3 || [http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] - CI (Lambda) Inverter
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|[[Image:PoPperflipping.png|350px]]
 +
| When a division of the cell happens, the recombinase system is activated. During this phase there is no physical active connection between the output region and a promoter, thus the PoPS flux is considered to be zero.
 +
| Not controlled, expression should be zero.
|-
|-
-
|Promoter 0 || Exp3, Final PoPper||  
+
|[[Image:PoPperbackward.png|350px]]
 +
| During this phase, the output of the device is controlled by promoter B. We assume there is a low Repressor B concentration at this time. Thus Promoter B generates a flux of PoPS output. After a certain amount of time, Repressor B accumulates, since the constitutive promoter P placed upstream controls its expression. When the concentration of Repressor B is sufficient, the flux of PoPS goes to zero. Meanwhile, the expression of Repressor A is not active. Thus the concentration of Repressor A should decrease to a low level.
 +
|Controlled by Promoter B.
|-
|-
-
|Promoter A || Final PoPper||
+
|}
 +
 
 +
==Implementation==
 +
 
 +
In the previous section we define which functional components are needed at the level of parts. In this section the members with biology competences evaluate the design and identify the actual biological parts that can be used. Further on, the DNA abstraction level details are proposed.
 +
 
 +
{| width="20%" align="right" style="text-align:center"
|-
|-
-
|Repressor (+RBS) A || Final PoPper||
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|'''Part abstraction level (Implementation)'''
|-
|-
-
|Central Terminator || Final PoPper||
+
|[[Image:partslevel.png|350px]] Implementation of the part structure in the Division PoPper device.
|}
|}
-
===Biobricks requiring construction===
 
-
{|style="width:40%; background-color:#fcaf3e;" cellpadding="10"  
+
===Part abstraction level===
-
!Part || For use in
+
 
 +
'''The Division PoPper parts are:'''
 +
 
 +
* two Dif recombinase sites (design: Rec).
 +
* Tet promoter (design: promoter A).
 +
* TetR repressor (design: repressor A).
 +
* CLambda promoter (design: promoter B).
 +
* CLambda repressor (design: repressor B).
 +
* Lac promoter (design: promoter P).
 +
 
 +
The Registry of Standard Parts contains already some of the elements we need, for the others we constructed and added their BioBricked version.
 +
 
 +
'''The Division PoPper parts registry entries are:'''
 +
 
 +
* two Dif recombinase sites: [http://partsregistry.org/Part:BBa_I742101 BBa_I742101],[http://partsregistry.org/Part:BBa_I742102 BBa_I742102]
 +
* Tet promoter:[http://partsregistry.org/Part:BBa_R0040 BBa_R0040]
 +
* TetR repressor:[http://partsregistry.org/Part:BBa_C0040 BBa_C0040]
 +
* CLambda promoter:[http://partsregistry.org/Part:BBa_R0051 BBa_R0051]
 +
* CLambda repressor:[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]
 +
* Lac promoter:[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]
 +
 
 +
Click on the links for details.
 +
 
 +
 
 +
'''The assumptions regarding the biological elements are:'''
 +
 
 +
* The Dif sites flip only once during each cell division
 +
* TetR and ClambdaR are produced and degrade faster than the cell cycle
 +
 
 +
For this device to work, ''dif'' site-enclosed DNA needs to flip only once per division. Like all recombination sites, ''dif'' sites are directional and bacteria use directly repeated ''dif'' sites to resolve genome dimers. Research shows that this resolution occurs only at septation, and we use this temporal control to inverse a plasmid-borne sequence to induce different functions. For more information look at the [[Edinburgh/DivisionPopper/References|Background]] section.
 +
 
 +
'''The Biology Processes:'''
 +
We used the Edinburgh Pathway Notation, a state transition graphical notation to express the biological processes of the Division PoPper. The details regarding the notations can be found in the paper
 +
[http://www-bm.ipk-gatersleben.de/stable/php/journal/articles/pdf/jib-36.pdf A Graphical Notation to Describe the Logical Interactions of Biological Pathways].
 +
 
 +
 
 +
{| border="1" cellpadding="20" cellspacing="0"
 +
!Diagram
 +
!Explanation
|-
|-
-
|Dif sites || Exp1, Exp2, Final PoPper
+
|[[Image:PoPperEPN.jpg|550px]]
 +
|This EPN Diagram explains which are the state configuration of the Division PoPper construct when a Reporter Protein is placed downstream the device. The Division Event state explains which state transitions occur when a division happens. The gray boxes represent the state (active, inactive) of the coding regions and promoter regions associated to TetR and LambaCR production. The white boxes represent the proteins.
 +
|-
 +
|}
 +
 
 +
 
 +
{| width="20%" align="right" style="text-align:center"
 +
|-
 +
|'''DNA strands configuration'''
 +
|-
 +
|[[Image:DNAstrands.png|350px]] This graph explain on which DNA strand the parts are placed and with which coding direction.
 +
|}
 +
 
 +
===DNA abstraction level===
 +
 
 +
In order for this construct to be implemented as BioBricks combined in the normal fashion, it was necessary for some of the components to be synthesised in inverse orientation. The final list of components for the sequence, in order, is as follows:
 +
*''lac'' promoter [http://partsregistry.org/Part:BBa_I14032 BBa_I14032]
 +
*''dif'' site in forward orienation, generated by us as [http://partsregistry.org/Part:BBa_I742101 BBa_I742101]. (physically generated as a BioBrick and deposited).
 +
*reverse version of lambda cI-controlled promoter [http://partsregistry.org/Part:BBa_R0051 BBa_R0051]: designed as [http://partsregistry.org/Part:BBa_I742126 BBa_I742126].
 +
*ribosome binding site [http://partsregistry.org/Part:BBa_B0034 BBa_B0034].
 +
*Tet repressor coding sequence with rapid degradation tag [http://partsregistry.org/Part:BBa_C0040 BBa_C0040].
 +
*bidirectional terminator [http://partsregistry.org/Part:BBa_B0011 BBa_B0011] (initially it was planned to use a combination of several terminators here in different orientations, but after consultation with DNA synthesis company GeneArt, it was considered that the repeated sequences would cause difficulties in synthesis. If termination appears to be insufficient, sub-regions of the device can be extracted as separate biobricks by PCR, and further terminators inserted). In the final design submited to GeneArt, for certain reasons an inverse version of BBa_B0011 was used, and this was entered in our sandbox as [http://partsregistry.org/Part:BBa_I742129 BBa_I742129].
 +
*Reverse coding sequence of lambda cI repressor BBa_C0051, entered in our sandbox as [http://partsregistry.org/Part:BBa_I742131 BBa_I742131].
 +
*Reverse ribosome binding site BBa_B0034, entered in our sandbox as [http://partsregistry.org/Part:BBa_I742130 BBa_I742130]. Since the normal abbreviated biobrick scar between RBS and coding sequence could not be used in the inverse orientation, the length of the RBS was decreased by two bases to maintain the proper spacing.
 +
*Tet repressor-controlled promoter [http://partsregistry.org/Part:BBa_R0040 BBa_R0040].
 +
*''dif'' site in reverse orientation, generated as [http://partsregistry.org/Part:BBa_I742102 BBa_I742102] and deposited.
 +
*The total construct has been entered in our sandbox as [http://partsregistry.org/Part:BBa_I742132 BBa_I742132].
 +
 
 +
In order for the device to be tested, it will be necessary to insert it into a '''single copy BioBrick vector''', since if multiple copies are present the likelihood is increased that not all will be in the same orientation, leading to simultaneous production of both repressors and lack of output (this cross-talk between separate instances of the device within a single system is a characteristic feature of biological systems which must always be considered) . For this purpose, the single copy F'-episome-based plasmid '''pHR277''' (kanR) was generously provided by Professor Millicent Masters, Institute of Cell Biology, University of Edinburgh. This plasmid possesses unique XbaI and PstI sites which can be used to insert a biobrick; unfortunately, it also possesses an EcoRI site elsewhere on the plasmid so, unless this is removed, it cannot be converted into a fully compliant vector which can be used to assemble new BioBricks (in contrast to our Gram positive BioBrick vector pTG262-BB (BBa_I742123), described elsewhere in this wiki).
 +
 
 +
==Proofs of Concept==
 +
 
 +
The complex structure of the Division PoPper and the small amount of time we have for the construction of the device in the context of the iGEM competition led us to plan the construction of a simpler construct: the Proof of Concept device. The aim of the Proof of Concept device is to  test the main assumptions at the basis of the Division PoPper device: that the recombinase mechanism is activated only once and at cell division and that the time between divisions is sufficient for the repression mechanism to be activated and inactivated.  We planned to construct two experiments:
 +
 
 +
'''Experiment 1:'''
 +
 
 +
[[Image:proof1backward.png|300px|float|right]]
 +
 
 +
This experiment will prove whether or not the DNA between the two ''dif'' sites flips at all during cell division. If the a flip occurs, then the direction the promoter operates will be changed and YFP will be expressed. The BioBrick used for YFP expression is [http://partsregistry.org/Part:BBa_E0430 BBa_E0430]. The reverse ''lac'' promoter has been constructed and deposited as [http://partsregistry.org/Part:BBa_I742124 BBa_I742124].
 +
 
 +
 
 +
'''Experiment 2:'''
 +
 
 +
[[Image:proof2forward.png|300px|float|right]]
 +
 
 +
This investigates the number of flips that occurs during division and will require rapidly degrading fluorescent proteins.
 +
After each division we expect to see a change in colour as the promoter activates a different reporter. We developed some mathematical models of this scenario that can be seen in the [[Edinburgh/DivisionPopper/Modelling|Modelling]] section. In the [[Edinburgh/DivisionPopper/Status|Wet Lab]] section you can find all the detail regarding the construction in the lab.
 +
 
 +
 
 +
{| border="1" cellpadding="20" cellspacing="0"
 +
!Diagram
 +
!Explanation
|-
|-
-
|Reversed Promoter B || Exp1, Exp3, Final PoPper
+
|[[Image:Proof2EPN.jpg|550px]]
 +
|This EPN Diagram explains which are the state configuration of the Proof of Concept construct with YFP and GFP production. The Division Event state explains which state transitions occur when a division happens. The grey boxes represent the state (active, inactive) of the genes associated to YFP and GFP production and the white boxes represent the two proteins.
|-
|-
-
|Reversed Repressor (+RBS)B || Final PoPper
 
|}
|}

Latest revision as of 20:50, 26 October 2007

MENU : Introduction | Background | Applications | Design&Implementation | Modelling | Wet Lab | Synthetic Biology Approach | Conclusions

In the Design section we define the architecture (design), the biology elements (implementation) and the mechanisms (dynamics) of the device. According to the [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi abstraction levels hierarchy], we propose a view of the Division PoPper at the level of device, parts and DNA. Regarding to the dynamics, we explain which biological processes are involved and how they cooperate in defining the behaviour of the device. The logic design and implementation phases have been decoupled.

Contents



Design

In this section we explain the functional components of the device and how they are connected logically and physically. This view of the device follows the [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi abstraction hierarchy] proposed in the Registry of Parts.


Device abstraction level of the Division Counter
Devicelevel.png The Division PoPper device generates an input in the form of PoPS signal.


Device abstraction level

The Division PoPper is a device, because it is a functional element that can be further combined with other devices by the use of a standard PoPS signal. The device has no formal input but its behaviour is triggered by an "external" stimulus: the cell division. The output is a PoPS signal that is supposed to be always zero (not considering leaking issues in the promoters) apart from the short period immediately subsequent to a cell division event. In this case the device generates a pulse: the PoPS signal reaches a non-zero level for a period of time, returning to zero afterward. The function of the device is simple: to inform a downstream device that a division has happened by sending a pulse signal. We suggest some system configurations in the Applications section.







Part abstraction level (Design)
Partslevel(logic).png Design of the part structure in the Division PoPper device.

Part abstraction level

At the level of part abstraction we define the functional parts of the device: regulatory regions, coding regions, terminators and so on. Instead of proposing directly the actual biological entities, we propose here a logic view. Strictly speaking, we define entities by their attributes and abilities, but without mapping them to real biological objects (his is done in the Implementation section below). The motivation is to separate the functional design from the realization choices.

The Division PoPper functional parts are:

  • two recombinase site sequences: Rec.
  • a promoter A that can be repressed by protein A: promoter A.
  • a coding region for protein A: repressor A.
  • a promoter B that can be repressed by protein B: promoter B.
  • a coding region for protein B: repressor B.
  • a promoter P that is constitutively active (expressing).


The functional ability that these entities should have are:

  • the region between Rec recombinase site sequence is inverted at each cell division.
  • the protein A and protein B degradation rates should be fast enough to degrade them rapidly when not expressed.
  • the protein A and protein B effects on (respectively) the promoter A and B expression capacity should be strong.
  • promoter P should be strong in its constitutive expression.


The physical configuration:

The design relies on the alternate expression of the two strands of the DNA segment in between the Recombinase sites. Promoter P is placed outside the "flipping region" and controls its downstream region in the upper strand. The output of the device is given by the PoPS activity on the upper strand downstream from the second recombinase site. Since the region in between the recombinase sites flips, the output is controlled alternatively by the two DNA strands. In one strand the parts sequence is: repressor A, terminator, Promoter A. In the other the sequence is: repressor B, terminator, Promoter B.


The dynamics:

As cell division occurs, DNA enclosed by two inverted Recombinase sites is inverted. Initially the device is in the configuration with Repressor A and Promoter A on the upper strand. There is no Repressor A present in the cell, so Promoter A produces a constant PoPS output. As time passes, Repressor A accumulates and 'turns off' Promoter B. Meanwhile Repressor B is no produced and degrades so that, at the next division, Promoter B can produce PoPS output and the process repeats. This mechanism can be seen as the sequence of three phases: Forward, Flipping and Backward. We explain for each phases the dynamics:

Configuration Processes Output
PoPperforward.png During this phase, the output of the device is controlled by Promoter A. We assume there is no Repressor A present initially. Thus Promoter A generates a flux of PoPS output. After a certain amount of time, Repressor A builds up, since the constitutive promoter P placed upstream controls is expression. When the concentration of Repressor A is high enough, the flux of PoPS from the output promoter goes to zero. Meanwhile, the expression of Repressor B is not active. Thus the concentration of Repressor B should decrease to a low level. Controlled by Promoter A.
PoPperflipping.png When a division of the cell happens, the recombinase system is activated. During this phase there is no physical active connection between the output region and a promoter, thus the PoPS flux is considered to be zero. Not controlled, expression should be zero.
PoPperbackward.png During this phase, the output of the device is controlled by promoter B. We assume there is a low Repressor B concentration at this time. Thus Promoter B generates a flux of PoPS output. After a certain amount of time, Repressor B accumulates, since the constitutive promoter P placed upstream controls its expression. When the concentration of Repressor B is sufficient, the flux of PoPS goes to zero. Meanwhile, the expression of Repressor A is not active. Thus the concentration of Repressor A should decrease to a low level. Controlled by Promoter B.

Implementation

In the previous section we define which functional components are needed at the level of parts. In this section the members with biology competences evaluate the design and identify the actual biological parts that can be used. Further on, the DNA abstraction level details are proposed.

Part abstraction level (Implementation)
Partslevel.png Implementation of the part structure in the Division PoPper device.


Part abstraction level

The Division PoPper parts are:

  • two Dif recombinase sites (design: Rec).
  • Tet promoter (design: promoter A).
  • TetR repressor (design: repressor A).
  • CLambda promoter (design: promoter B).
  • CLambda repressor (design: repressor B).
  • Lac promoter (design: promoter P).

The Registry of Standard Parts contains already some of the elements we need, for the others we constructed and added their BioBricked version.

The Division PoPper parts registry entries are:

  • two Dif recombinase sites: [http://partsregistry.org/Part:BBa_I742101 BBa_I742101],[http://partsregistry.org/Part:BBa_I742102 BBa_I742102]
  • Tet promoter:[http://partsregistry.org/Part:BBa_R0040 BBa_R0040]
  • TetR repressor:[http://partsregistry.org/Part:BBa_C0040 BBa_C0040]
  • CLambda promoter:[http://partsregistry.org/Part:BBa_R0051 BBa_R0051]
  • CLambda repressor:[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]
  • Lac promoter:[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]

Click on the links for details.


The assumptions regarding the biological elements are:

  • The Dif sites flip only once during each cell division
  • TetR and ClambdaR are produced and degrade faster than the cell cycle

For this device to work, dif site-enclosed DNA needs to flip only once per division. Like all recombination sites, dif sites are directional and bacteria use directly repeated dif sites to resolve genome dimers. Research shows that this resolution occurs only at septation, and we use this temporal control to inverse a plasmid-borne sequence to induce different functions. For more information look at the Background section.

The Biology Processes: We used the Edinburgh Pathway Notation, a state transition graphical notation to express the biological processes of the Division PoPper. The details regarding the notations can be found in the paper [http://www-bm.ipk-gatersleben.de/stable/php/journal/articles/pdf/jib-36.pdf A Graphical Notation to Describe the Logical Interactions of Biological Pathways].


Diagram Explanation
PoPperEPN.jpg This EPN Diagram explains which are the state configuration of the Division PoPper construct when a Reporter Protein is placed downstream the device. The Division Event state explains which state transitions occur when a division happens. The gray boxes represent the state (active, inactive) of the coding regions and promoter regions associated to TetR and LambaCR production. The white boxes represent the proteins.


DNA strands configuration
DNAstrands.png This graph explain on which DNA strand the parts are placed and with which coding direction.

DNA abstraction level

In order for this construct to be implemented as BioBricks combined in the normal fashion, it was necessary for some of the components to be synthesised in inverse orientation. The final list of components for the sequence, in order, is as follows:

  • lac promoter [http://partsregistry.org/Part:BBa_I14032 BBa_I14032]
  • dif site in forward orienation, generated by us as [http://partsregistry.org/Part:BBa_I742101 BBa_I742101]. (physically generated as a BioBrick and deposited).
  • reverse version of lambda cI-controlled promoter [http://partsregistry.org/Part:BBa_R0051 BBa_R0051]: designed as [http://partsregistry.org/Part:BBa_I742126 BBa_I742126].
  • ribosome binding site [http://partsregistry.org/Part:BBa_B0034 BBa_B0034].
  • Tet repressor coding sequence with rapid degradation tag [http://partsregistry.org/Part:BBa_C0040 BBa_C0040].
  • bidirectional terminator [http://partsregistry.org/Part:BBa_B0011 BBa_B0011] (initially it was planned to use a combination of several terminators here in different orientations, but after consultation with DNA synthesis company GeneArt, it was considered that the repeated sequences would cause difficulties in synthesis. If termination appears to be insufficient, sub-regions of the device can be extracted as separate biobricks by PCR, and further terminators inserted). In the final design submited to GeneArt, for certain reasons an inverse version of BBa_B0011 was used, and this was entered in our sandbox as [http://partsregistry.org/Part:BBa_I742129 BBa_I742129].
  • Reverse coding sequence of lambda cI repressor BBa_C0051, entered in our sandbox as [http://partsregistry.org/Part:BBa_I742131 BBa_I742131].
  • Reverse ribosome binding site BBa_B0034, entered in our sandbox as [http://partsregistry.org/Part:BBa_I742130 BBa_I742130]. Since the normal abbreviated biobrick scar between RBS and coding sequence could not be used in the inverse orientation, the length of the RBS was decreased by two bases to maintain the proper spacing.
  • Tet repressor-controlled promoter [http://partsregistry.org/Part:BBa_R0040 BBa_R0040].
  • dif site in reverse orientation, generated as [http://partsregistry.org/Part:BBa_I742102 BBa_I742102] and deposited.
  • The total construct has been entered in our sandbox as [http://partsregistry.org/Part:BBa_I742132 BBa_I742132].

In order for the device to be tested, it will be necessary to insert it into a single copy BioBrick vector, since if multiple copies are present the likelihood is increased that not all will be in the same orientation, leading to simultaneous production of both repressors and lack of output (this cross-talk between separate instances of the device within a single system is a characteristic feature of biological systems which must always be considered) . For this purpose, the single copy F'-episome-based plasmid pHR277 (kanR) was generously provided by Professor Millicent Masters, Institute of Cell Biology, University of Edinburgh. This plasmid possesses unique XbaI and PstI sites which can be used to insert a biobrick; unfortunately, it also possesses an EcoRI site elsewhere on the plasmid so, unless this is removed, it cannot be converted into a fully compliant vector which can be used to assemble new BioBricks (in contrast to our Gram positive BioBrick vector pTG262-BB (BBa_I742123), described elsewhere in this wiki).

Proofs of Concept

The complex structure of the Division PoPper and the small amount of time we have for the construction of the device in the context of the iGEM competition led us to plan the construction of a simpler construct: the Proof of Concept device. The aim of the Proof of Concept device is to test the main assumptions at the basis of the Division PoPper device: that the recombinase mechanism is activated only once and at cell division and that the time between divisions is sufficient for the repression mechanism to be activated and inactivated. We planned to construct two experiments:

Experiment 1:

float

This experiment will prove whether or not the DNA between the two dif sites flips at all during cell division. If the a flip occurs, then the direction the promoter operates will be changed and YFP will be expressed. The BioBrick used for YFP expression is [http://partsregistry.org/Part:BBa_E0430 BBa_E0430]. The reverse lac promoter has been constructed and deposited as [http://partsregistry.org/Part:BBa_I742124 BBa_I742124].


Experiment 2:

float

This investigates the number of flips that occurs during division and will require rapidly degrading fluorescent proteins. After each division we expect to see a change in colour as the promoter activates a different reporter. We developed some mathematical models of this scenario that can be seen in the Modelling section. In the Wet Lab section you can find all the detail regarding the construction in the lab.


Diagram Explanation
Proof2EPN.jpg This EPN Diagram explains which are the state configuration of the Proof of Concept construct with YFP and GFP production. The Division Event state explains which state transitions occur when a division happens. The grey boxes represent the state (active, inactive) of the genes associated to YFP and GFP production and the white boxes represent the two proteins.
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