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Revision as of 05:39, 14 August 2012

Background

Bacteriophytochrome

Phytochromes are light receptor proteins that are used in order to sense the presence, intensity, duration, direction of light. They are prevalent in plants as well as cyanobacteria and are used to control morphological developments. These developments include seed germination, flower generation, responses to sunlight. Phytochromes are able to absorb light in the red (620-750 nm) and far-red region (700-800 nm).

A slightly modified version of the phytochrome is also present in bacteria. The aptly named bacteriophytochrome is present in photosynthetic and nonphotosynthetic. Bacteriophytochromes from Agrobacterium tumefaciens and Deinococcus radiodurans contain the structural motif: PAS-GAF-PHY-HisKinase. The PAS domain contains a chromophore binding site where biliverdin can bind and harness light. The GAF domain allows for attachment of the chromaphore and the PHY domain contributes along with PAS to help the chromaphore adopt the proper conformation.

Biliverdin is the product of oxidation of heme B and is catalysed by heme oxygenase. This enzyme is not native to E. coli and so needs to be introduced in the bacteriophytochrome vector. A Cys residue in the bacteriophytochrome binds the biliverdin within the protein.

Bacteriophytochromes are able to alternate between the two distinct forms with different activities. The phytochrome naturally exists in the inactive state (Pr) where it can absorb red light and if the cell is overproducing the protein it appears blue. After it absorbs red light it undergoes a conformation change to the far-red state (Pfr). This is known as the active form, and makes the cell appear green. When, the cell is irradiated with far-red light then it is able to isomerize back to the Pr form.

In this way we produce an inducible switch and are able to control the colour of E. coli. If the cell is irradiated with red light and emits a green colour, hit it with far red light and emits a blue colour. The switch is controlled simply by light and due to this there is significant potential for differential expression of two different kinase pathways. There is also the advantage of a clear detection mechanism, which would be useful as a reported gene.

The bacteriophytochromes of Agrobacterium tumefaciens and Deinococcus radiodurans are able to be excited by two different wavelengths of red light. So if we subject E. coli with different wavelengths of light we can activate different biochemical pathways.

The bacteriophytochrome vector will be transformed into E. coli competent BL21 (DE3) cells, which contains the T7 RNA polyermase for the expression of T7 promoter regulated operons. As E. coli do not contain a native heme oxygenase gene, it must be incorporated into the final assembled operon construct along with a T7 promoter and the bacteriophytochrome genes from Deinococcus and Agrobacterium for the "light switch" to be self-assembled and functional

Gibson Cloning

Gibson cloning is a powerful and innovative method for cloning a gene into an expression vector.