CH391L/S12/Pattern Formation

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(New page: =Synthetic Pattern Formation= Pattern formation is a ubiquitous feature of biology. Organisms have evolved to receive signals, transduce the message, and elicit an appropriate response. Pa...)
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==Synthetic Pattern Formation Driven by Light==
==Synthetic Pattern Formation Driven by Light==
The synthetic biology community has been using light as a tool for pattern formation in microbes since 2002. <cite>shimizu2002</cite> Light has been utilized for pattern formation in ''E. coli'' for the first ever organismal photograph<cite>Levskaya</cite>. Since then that system was modified to be an "edge detector" of light<cite>Tabor2009</cite>. The focus of this section will be on the paper ''Multichromatic Control of Gene Expression in Escherichia coli''<cite>Tabor2011</cite>.  
The synthetic biology community has been using light as a tool for pattern formation in microbes since 2002. <cite>shimizu2002</cite> Light has been utilized for pattern formation in ''E. coli'' for the first ever organismal photograph<cite>Levskaya</cite>. Since then that system was modified to be an "edge detector" of light<cite>Tabor2009</cite>. The focus of this section will be on the paper ''Multichromatic Control of Gene Expression in Escherichia coli''<cite>Tabor2011</cite>.  
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Having multiple light induction systems is great for having high resolution spatio-temporal control of gene expression.This paper essentially describes a two wavelength (RED and GREEN) gene expression system.  Essentially, gene expression can be turned on via RED light or GREEN light in a specific and orthogonal manner. The RED system was borrowed from the initial photograph forming E.coli. Briefly, the RED system uses an engineered phytochrome, cph8 ,coupled to a EnvZ/OmpR pathway to activate gene expression. In the gene circuit cph8 is produced in the phosphorylated "ON" state, which upon induction of RED light switches to the unphosphorylated "OFF" state. In order for the RED light to produce LacZ, the cph8 regulates an inhibitor (C1) that binds to the LacZ reporter.  In the dark cph8 is "ON" which will make C1 and keep the LacZ "OFF." But when RED light is present the cph8 no longer expresses the C1, thus allowing LacZ production. The cph8 system can be considered a NOT gate, or genetic inverter.
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The RED system was tested by the Miller assay (a quantitative LacZ expression assay). In Dark conditions 0.58MU were produced as compared to  RED conditions 1.41MU. This corresponds to an induction of 2.4 fold, which is similar to the green system. The RED system is dependent on the PCB chromophore, which is produced from heme by two genes on a plasmid. For proper induction under the corresponding light-wavelengths, transfer functions of induction were tested. This essentially tests the induction of LacZ expression across the spectrum of light. As it turns out, the RED system is turned on partially at the Green induction wavelength (532nM).
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The GREEN system is a two component system involving a membrane associated histidine kinase CcaS and response regulator CcaR. Green light increases the rate of CcaS autophosphorylation, phosphotransfer to CcaR, and transcription by promoter cpcG2 upon CcaR binding. This is different than the RED system, which turns expression OFF in Red light, because it directly turns ON gene expression under green light conditions.

Revision as of 02:21, 2 April 2012

Synthetic Pattern Formation

Pattern formation is a ubiquitous feature of biology. Organisms have evolved to receive signals, transduce the message, and elicit an appropriate response. Patterns are formed by cells deferentially responding to signals, whether it be from signals generated from other cells or external features such as light. In recent years, biologists are beginning to create synthetic pattern formation algorithms which allow for manipulation of biology on a more complex level. This page will specifically focus on synthetic pattern formation in E.coli.

Synthetic Pattern Formation Driven by Light

The synthetic biology community has been using light as a tool for pattern formation in microbes since 2002. [1] Light has been utilized for pattern formation in E. coli for the first ever organismal photograph[2]. Since then that system was modified to be an "edge detector" of light[3]. The focus of this section will be on the paper Multichromatic Control of Gene Expression in Escherichia coli[4].

Having multiple light induction systems is great for having high resolution spatio-temporal control of gene expression.This paper essentially describes a two wavelength (RED and GREEN) gene expression system. Essentially, gene expression can be turned on via RED light or GREEN light in a specific and orthogonal manner. The RED system was borrowed from the initial photograph forming E.coli. Briefly, the RED system uses an engineered phytochrome, cph8 ,coupled to a EnvZ/OmpR pathway to activate gene expression. In the gene circuit cph8 is produced in the phosphorylated "ON" state, which upon induction of RED light switches to the unphosphorylated "OFF" state. In order for the RED light to produce LacZ, the cph8 regulates an inhibitor (C1) that binds to the LacZ reporter. In the dark cph8 is "ON" which will make C1 and keep the LacZ "OFF." But when RED light is present the cph8 no longer expresses the C1, thus allowing LacZ production. The cph8 system can be considered a NOT gate, or genetic inverter.

The RED system was tested by the Miller assay (a quantitative LacZ expression assay). In Dark conditions 0.58MU were produced as compared to RED conditions 1.41MU. This corresponds to an induction of 2.4 fold, which is similar to the green system. The RED system is dependent on the PCB chromophore, which is produced from heme by two genes on a plasmid. For proper induction under the corresponding light-wavelengths, transfer functions of induction were tested. This essentially tests the induction of LacZ expression across the spectrum of light. As it turns out, the RED system is turned on partially at the Green induction wavelength (532nM).

The GREEN system is a two component system involving a membrane associated histidine kinase CcaS and response regulator CcaR. Green light increases the rate of CcaS autophosphorylation, phosphotransfer to CcaR, and transcription by promoter cpcG2 upon CcaR binding. This is different than the RED system, which turns expression OFF in Red light, because it directly turns ON gene expression under green light conditions.








References

  1. PMID: 16306980 [Levskaya]
    Engineering E. coli to see Light

  2. PMID: 19563759 [Tabor2009]
    Edge Detection Program

  3. PMID: 21035461 [Tabor2011]
    Multichromatic Control of gene expression

  4. PMID: 12219076 [Shimizu2002]
    Light Switchable System

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