Edinburgh/DivisionPopper

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Latest revision as of 01:38, 27 October 2007

The Division PoPper is a signal generator device that produces an output of [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi PoPS] as a function of bacterial cell division. In simple terms, it is a device that generates a "pulse" of PoPS signal each time a cell undertakes division. Downstream of the device may be a counter device, quantifiable protein production or some other function of choice. The system may for example perform pre-determined actions such as programmed cell death after a set number of cell divisions or being used to calculate the division frequency. The device relies on dif recombinase sites to flip a DNA segment at each cell division. With this project we hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood. We plan to construct and ligate the bricks required for a first proof of concept experiments. We model the device using various approaches (deterministic and stochastic modelling) in order to guide lab work and analyse lab data. Modelling is also used to simulate the composition of our device with a counter device. In presenting our work, we highlight with comments and discussion the application of Synthetic Biology approaches at each work phase.

Division PoPper device

The Division PoPper device generates a pulse of PoPS signal at each cell division.

Sections:

The structure of the wiki for this project is composed of:

This Introduction page has a brief overview.
The Applications page gives some possible uses of the Division PoPper.
The Background page explains the scientific background of our project and lists paper references validating our work.
The Design & Implementation page explains the architecture and the mechanisms of the device in terms of abstraction levels and biological processes.
The Modelling page contains the computational and mathematical investigation of the system behaviour.
The Wet Lab page reports our achievements, so far, in the wet lab.
The section Synthetic Biology Approach discusses how we applied the Synthetic Biology approach to the project construction.
The Conclusions page summarizes our final consideration of the project.

A menu with the same links is present at the top of each page for facilitating navigation.

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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]]
-== POPS Oscillator reporting cell division ==
-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''' Division PoPper''' is a signal generator device that produces an output of [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi PoPS] as a function of bacterial cell division. In simple terms, it is a device that generates a "pulse" of PoPS signal each time a cell undertakes division. Downstream of the device may be a counter device, quantifiable protein production or some other function of choice. The system may for example perform pre-determined actions such as programmed cell death after a set number of cell divisions or being used to calculate the division frequency.
+The device relies on dif recombinase sites to flip a DNA segment at each cell division. With this project we hope to further analyse cell division and recombinase mechanisms since bacterial cell division is still relatively poorly understood. We plan to construct and ligate the bricks required for a first proof of concept experiments. We model the device using various approaches (deterministic and stochastic modelling) in order to guide lab work and analyse lab data. Modelling is also used to simulate the composition of our device with a counter device.
+In presenting our work, we highlight with comments and discussion the application of Synthetic Biology approaches at each work phase.
-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===
+{| width="20%" align="right" style="text-align:center"
+|-
+|'''Division PoPper device'''
+|-
+|[[Image:Overview.png|500px]] The Division PoPper device generates a pulse of PoPS signal at each cell division.
+|}
-[[Image:Division pulser.png|800px]]
+===Sections:===
+The structure of the wiki for this project is composed of:
-This device outputs a PoPS pulse at each cell division.
+* This [[Edinburgh/DivisionPopper| '''Introduction''']] page has a brief overview.
-It can then be hooked up to another device such as a counter.
+* The [[Edinburgh/DivisionPopper/Applications|'''Applications''']] page gives some possible uses of the Division PoPper.
+* The [[Edinburgh/DivisionPopper/References|'''Background''']] page explains the scientific background of our project and lists paper references validating our work.
+* The [[Edinburgh/DivisionPopper/Design|'''Design & Implementation''']] page explains the architecture and the mechanisms of the device in terms of abstraction levels and biological processes.
+* The [[Edinburgh/DivisionPopper/Modelling|'''Modelling''']] page contains the computational and mathematical investigation of the system behaviour.
+* The [[Edinburgh/DivisionPopper/Status|'''Wet Lab''']] page reports our achievements, so far, in the wet lab.
+* The section '''[[Edinburgh/DivisionPopper/SBApproach|Synthetic Biology Approach]]''' discusses how we applied the Synthetic Biology approach to the project construction.
+* The [[Edinburgh/DivisionPopper/Conclusions|'''Conclusions''']] page summarizes our final consideration of the project.
-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, <font color="blue">'''Promoter A'''</font> is repressed by continually produced <font color="blue">'''represser A'''</font>. At this time, no <font color="orange">'''represser B'''</font> is produced.
-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 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====
-[[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.
-We might wish to test the repressors without the dif sites
-[[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:
-* 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]]
-'''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
-===References===
-====[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1449051&blobtype=pdf Bloor]====
-- An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes (2006)
-: This article describes a system where a gene is deleted by replacing it with an antibiotic marker. The antibiotic marker is then excised during next cell divsion using the naturally expressed XerCD? recombinases. This is similar to what we are doing except instead of excising we are inverting.
-{|align="center" style="width:100%; border:2px #a3b1bf solid; background:#f5faff; text-align:left;"
-|-
-|"The natural dif site is in the chromosome terminus region and is essential for correct chromosome segregation at cell division: deletion of difE. coli results in a subpopulation of E. coli cells with a filamentous morphology (12). However, if natural difE. coli is deleted, another difE. coli site will not enable dimer resolution if placed outside of the  30-kb dif activity zone (DAZ) in the terminus region (6, 16). Therefore, while our method involves insertion of one or more extra dif sites at different chromosomal loci, this should not have an adverse effect on chromosome segregation, although the possibility that introduction of multiple dif sites in close proximity might result in deletion of intervening chromosomal DNA should be considered."
-|}
-:: '''Summary:''' Dif sites outside of the DAZ zone shouldn't cause problems with the natural dif sites of the chromosome/plasmid
-====[http://www.blackwell-synergy.com/doi/full/10.1046/j.1365-2958.1998.00651.x Kuempel]====
-- Cell division is required for resolution of dimer chromosomes at the dif locus of Escherichia coli (1998)
-: Good overview of the natural Xer recombinase system.
+<center>
+''A menu with the same links is present at the top of each page for facilitating navigation.''

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Edinburgh/DivisionPopper

From 2007.igem.org

Latest revision as of 01:38, 27 October 2007

Sections: