Memory effects of UV exposure

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-		+	We have placed the lacY gene under the SOS (UV responsive) promoter. Since the production of Lac Y and the effect of build of Lac Y upon our memory switch would take time, we needed an  immediate reporter of promoter activity . So the mRFP gene was also placed under the SOS promoter to make the final part - mRFP and LacY gene under the SOS promoter.
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	===Testing Bisability in the UV-switch strain===		===Testing Bisability in the UV-switch strain===

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Revision as of 05:41, 31 October 2006

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Contents 1 Group III 1.1 Aim 1.2 Approach 1.3 Constructs 1.4 Testing Bisability in the UV-switch strain

Group III

Aim

We are interested in converting a transient UV exposure into a persistant response. We plan to achieve this by coupling UV response system to a bistable network. Such system can be used to monitor cells that have undergone UV damage.

Approach

So what is this UV damage response and why long term monitoring?

The SOS response

The SOS genetic network consists of various genes that perform diverse functions in response to DNA Damage NER (Nucleotide Excision Repair) Translesion DNA replication Homologous recombination Cell division arrest

Lex A is the master repressor which binds sites in the promoter regions of the genes which are part of the SOS response

RecA acts as a sensor of DNA Damage RecA gets activated and mediates LexA auto-cleavage

Salient features of the SOS response

Transient response induced in response to DNA damage. The promoter activity increases rapidly in the first few minutes and decrease within an hour.

Thus, long time monitoring of DNA Damage induced by UV Irradiation requires that this transient signal generated is stabilized.

So, we tried to couple this transient signal to a bistable network to enable long time monitoring of DNA Damage.

We have used the lac regulatory network as the bistable network.

Constructs

Here comes the exciting stuff, the synthetic biology part! We have made the following parts

We have placed the lacY gene under the SOS (UV responsive) promoter. Since the production of Lac Y and the effect of build of Lac Y upon our memory switch would take time, we needed an immediate reporter of promoter activity . So the mRFP gene was also placed under the SOS promoter to make the final part - mRFP and LacY gene under the SOS promoter.

Testing Bisability in the UV-switch strain

The regulatory network involved in uptake and utilization of lactose exhibits bistability over a range environmental conditions [Ozbudak et al., 2004]. This has been demonstrated in an E.coli strain Muk21 that has chromosomal integration of GFP expressed under lac promoter.

A bistable network is hysteretic. Environmental history determines state of the network at intermediate inducer concentration.

This is a scatter plot of cells. Cells with uninduced (lower panel) or induced (upper panel) history are grown in different TMG concentrations.

We transformed Muk21 with our UV-switch construct to test hysteresis which is a characteristic behavior of a bistable network.

This is a scatter plot of cells. Cells with uninduced (;eft panel) or induced (right panel) history are grown in different TMG concentrations.