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-	[https://2007.igem.org/Edinburgh https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG]	+	'''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 Research In it's current configuration, <font color="blue">'''Promoter A'''</font> is repressed by <font color="blue">'''represser A'''</font> which is continually being produced. At this time there is no <font color="orange">'''represser B'''</font> being produced.
-	When cell division occurs, the section of DNA between the two dif sites gets reversed. Initially there is no <font color="orange">'''represser B'''</font> present in the cell so <font color="orange">'''promoter B'''</font> is able to produce an output of PoPS. As time goes on <font color="orange">'''represser B'''</font> gets produced and 'turns off' <font color="orange">'''promoter B'''</font>. At the same time <font color="blue">'''represser A'''</font> is no longer being produced and degrades so when the next division occurs, <font color="blue">'''promoter A'''</font> will be capable of producing a 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 the production of represser 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 will prove whether or not it flips once during division, this will require fast degrading fluorescent proteins.
-
-	After each division we expect to see a change in colour as the promoter is activating a separate gene
-
-	====Exp 3====
-
-	Currently we are planning to test how the dif sites flip using exps 1 & 2 however there is still a large
-	jump to the final device.
-
-	It was suggested we should test the represser part of the system without the dif sites
-
-	[[Image:Edinburgh_Division_pulser.invert.png|500px]]
-
-	The represser 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 promoting represer A and tests  the standard represser 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 – suggest 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)]====	+	<center>
-	: Good overview of the natural Xer recombinase system.	+	''A menu with the same links is present at the top of each page for facilitating navigation.''

---

Latest revision as of 01:38, 27 October 2007

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

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.