From 2011.igem.org

Revision as of 09:18, 28 September 2011

ITESM MÉXICO

SensE.coli

Modeling -_ Conentration and photoreceptor

Basic steps:

- Transformation of competent cells with iGEM parts and our own designed parts

- Plasmid isolation of each part

- Run an electrophoresis gel to detect the presence of DNA

- Purification of gel bands to get the parts of interest

- Digestion of the pieces with EcoRI, XbaI, SpeI, PstI enzymes

- Run electrophoresis gel to detect the digestion products

- Purification of the pieces from the gel

- Ligation of the pieces

- Repeat of the process of digestion and ligation to put together the whole construct

- Confirmatory test

Experimental Dessign:

Photoreceptor mechanism was used from the the Tokio-Nokogen team of 2009 (https://2009.igem.org/Team:Tokyo-Nokogen) This mechanism was modified by adding only the green light receptor instead of the whole mechanism., including also the red light receptor mechanism.

The most important modification of this mechanism was the inclusion of the protein RecA in our construct to make it compatible to the regulation system of lambda phage incorporated in the mechanism of concentration.

Green receptor: Is going to be used to initiate the entire mechanism. It is composed by eight parts, in a sequence of twelve parts.

· The constitutive promoter is used for the permanent expression of the gene, it is attached to a ribosome binding site (RBS) that is a sequence on the messenger RNA to which the ribosome can bind and initiate protein translation and produce the ho1 (heme oxigenase 1) that converts heme to biliverdin IXalpha.

· The sequence that produce pcy4A (phycocyanobilinàferredoxin oxidoreductase) allows the convertion of the biliverdin IXalpha to phycocyanobilin.

· Another RBS is used to produce CcaS-EnvZ which is a chimeric protein of ScaS and the EnvZ that acts as the light responsive domain. CcaS-EnvZ is the coding sequence of the green light receptor, it induces the expression of phycobilisome linker protein.

· The green light OmpR regulates the production of outer membrane proteins. This promoter will allow the expression of the RecA protein that activates the expression of the promoters for arabinose concentration by reducing the activity of the lambda repressor.

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RecA is a protein used for the cleavage of protein lambda. It has shown that it has a cleavage activity when its binded to lambda repressor. This is a essential part of the project, because these interactions are the link between the photoreceptor and the concentration mechanims by the lambda repressor and the lambda operators. (Sauer et al, 1981)

Concentration:

The concentration mechanism is based in the experiments and mechanisms developed by British Columbia University iGEM team in 2009 ( https://2009.igem.org/Team:British_Columbia). This is the part we focused on improving from the original idea. Basically the experiment was the same, there are only few changes from the original construct, like the addition of an anti-sense key to stop the action of the key that disables the capability of the reporter protein to express.

The construct is very similar to the one from UBC. We took the pieces Pbad st for sensing low concentrations and Pbad wk for sensing high concentrations. We re-designed the activation/inactivation (lock and key) based on the experiments of lock and key developed by UBC in 2009 and the control of gene expression (Isaacs et al,2004) · We re-designed some pieces to make them more specific, we modified the lock and key mechanism, and added more parts, one lock and key specific for the high concentration and other lock and key designed for low concentrations. Also the inclusion of one new part that regulates one of the keys by inactivating it.

The first part of the second construct consists in the constitutive promoter that produces the lambda repressor protein. This lambda repressor binds to the operators, inhibiting the expression of the mechanisms of concentration expression. The operators interacts with the repressor lambda inhibiting the action of the RNA polymerase to transcribe the sequence.

The next link is between the concentration mechanism and the lambda repressor. Those are the operators, that can be found before the promoter Pbad st. these operators are responsible for the binding of lambda repressor protein and for the expression of the reporter protein. If the operators are free then there can be expression. Operators can only be free once the RecA protein cleavages the lambda repressor, so the expression can be continued. Then the Pbad st can activate the expression of the reporter protein GFP, but there is a previous step where there is the first lock (crxst); This first lock inhibits the expression of the reporter protein and that’s why at the same concentration with the same promoter there is the activation of our key (tast). For high concentrations there is the promoter pbad wk, which can sense big quantities of arabinose. The promoter is also activated by action of the operators. Once the promoter is activated the process of expression is continued until the CFP is expressed. To express this protein the lock (crxwk) must be inactivated by the key (tawk). To assure that only the high concentration mechanism is enabled there is the necessity to turn down the low concentration, this is achieved by expressing an antisense key (itast) that inhibits the production of the low concentration key (tast) thus the low concentration lock (crxst) will activate and will inhibit the expression of GFP.