Edinburgh/DivisionPopper
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[https://2007.igem.org/Edinburgh https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG] | [https://2007.igem.org/Edinburgh https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG] | ||
| - | + | =Division PoPer= | |
| - | + | __TOC__ | |
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| + | ==Project Description== | ||
The aim is to create a signal generator device that produces an output of PoPS as a function of bacterial cell division. Down stream of the device there may be a counter device or a frequency analyser. | The aim is to create a signal generator device that produces an output of PoPS as a function of bacterial cell division. Down stream of the device there may be a counter device or a frequency analyser. | ||
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The device relies on using dif recombinase sites to flip once during each cell division. We hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood. | The device relies on using dif recombinase sites to flip once during each cell division. We hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood. | ||
| - | + | ==Current Device Design== | |
[[Image:Division pulser.png|800px]] | [[Image:Division pulser.png|800px]] | ||
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As cell division occurs, DNA enclosed by two inverted dif sites is reversed. Initially there is no <font color="orange">'''represser B'''</font> present in the cell so <font color="orange">'''promoter B'''</font> produces PoPS output. As time passes, <font color="orange">'''represser B'''</font> is produced and 'turns off' <font color="orange">'''promoter B'''</font>. Meanwhile <font color="blue">'''represser 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. | As cell division occurs, DNA enclosed by two inverted dif sites is reversed. Initially there is no <font color="orange">'''represser B'''</font> present in the cell so <font color="orange">'''promoter B'''</font> produces PoPS output. As time passes, <font color="orange">'''represser B'''</font> is produced and 'turns off' <font color="orange">'''promoter B'''</font>. Meanwhile <font color="blue">'''represser 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. | ||
| - | + | ==Assumptions== | |
* The Dif sites flip only once during each cell division | * The Dif sites flip only once during each cell division | ||
* Repressors A and B are produced and degrade faster than the cell cycle | * Repressors A and B are produced and degrade faster than the cell cycle | ||
* Promoter B does not interfere with production of repressor A | * Promoter B does not interfere with production of repressor A | ||
| - | + | ==Initial Experiments== | |
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. | 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]] | [[Image:Divisiontest1.png|300px|float|right]] | ||
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| - | + | ===Exp 2=== | |
[[Image:Edinburgh_Divisiontest2.png|300px|float|left]] | [[Image:Edinburgh_Divisiontest2.png|300px|float|left]] | ||
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After each division we expect to see a change in colour as the promoter activates a different reporter. | 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. | 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. | ||
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* Reporter – use same as experiment 1 | * Reporter – use same as experiment 1 | ||
| - | + | ==Biobricks Required== | |
| - | + | ===Biobricks From Registry=== | |
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| - | + | ===Biobricks requiring construction=== | |
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| - | + | ==References== | |
see [[Edinburgh/DivisionPopper/References]] | see [[Edinburgh/DivisionPopper/References]] | ||
Revision as of 10:18, 2 August 2007
https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG
Division PoPer
Contents |
Project Description
The aim is to create a signal generator device that produces an output of PoPS as a function of bacterial cell division. Down stream of the device there may be a counter device or a frequency analyser.
The system may for example perform pre-determined actions such as programmed cell death after a set number of cell divisions.
The device relies on using dif recombinase sites to flip once during each cell division. We hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood.
Current Device Design
This device outputs a PoPS pulse at each cell division. It can then 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 differential functions. In it's current configuration, Promoter A is repressed by continually produced represser A. At this time, no represser B is produced.
As cell division occurs, DNA enclosed by two inverted dif sites is reversed. Initially there is no represser B present in the cell so promoter B produces PoPS output. As time passes, represser B is produced and 'turns off' promoter B. Meanwhile represser A is no longer produced and degrades so that, at the next division, promoter A can produce PoPS output and the process repeats.
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
Initial Experiments
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
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
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.
We might wish to test the repressors without the dif sites
The repressor part of the system is simply a pair of inverters.
There are 3 basic types of inverter in the registry:
- 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]”
Suggested exp 3
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:
- Promoter 0 – suggest BBa_I0500 (arabinose) – Plate 2, 9I
- Promoter B – backward lacI – Used in exp 1
- Inverter – suggested BBa_Q04510 (CI (Lambda)) – Plate 2, 13K
- Reporter – use same as experiment 1
Biobricks Required
Biobricks From Registry
| Part | For use in | Brick used |
|---|---|---|
| Reporter | Exp1, Exp2, Exp3 | [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] - Fast Degrading ECFP |
| Inverter | Exp3 | [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] - CI (Lambda) Inverter |
| Promoter 0 | Exp3, Final PoPper | |
| Promoter A | Final PoPper | |
| Represer (+RBS) A | Final PoPper | |
| Central Termenator | Final PoPper |
Biobricks requiring construction
| Part | For use in |
|---|---|
| Dif sites | Exp1, Exp2, Final PoPper |
| Reversed Promoter B | Exp1, Exp3, Final PoPper |
| Represer (+RBS)B | Final PoPper |
References
see Edinburgh/DivisionPopper/References
