From 2007.igem.org

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Contents 1 Division PoPper 1.1 Project Goal 1.2 Current Device Idea 1.3 Assumptions 1.4 Initial Experiments 1.4.1 Exp 1 1.4.2 Exp 2 1.4.3 Exp 3 1.5 Biobricks Required 1.5.1 Biobricks From Registry 1.5.2 Biobricks requiring construction 1.6 References

Division PoPper

The idea is to use the XerC and D recombinases to invert a section of DNA at cell division. Inversion of the DNA causes a PoPS pulse as output.

Project Goal

The aim is to produce output as a function of bacterial cell division. Different versions of the recombination enable different downstream outputs. The system may for example perform programmed cell death after a set number of cell divisions. We hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood.

Current Device Idea

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 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	Exp3, Final PoPper
Represer (+RBS)B	Final PoPper

References

see Edinburgh/DivisionPopper/References