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Background

Bacteriophytochrome

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

The aptly named bacteriophytochrome is present in photosynthetic and nonphotosynthetic bacteria alike. Bacteriophytochromes from Agrobacterium tumefaciens and Deinococcus radiodurans contain the general structure with a N-terminal harvesting domain and C-terminal stimulatory domain. There is 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 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 conformational 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 isomerise back to the Pr form.

In this way we produce a reversible switch that enables the control of the colour of E. coli. If the cell is irradiated with red light it 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 reporter gene.

The bacteriophytochromes of Agrobacterium tumefaciens and Deinococcus radiodurans are able to be excited by two different wavelengths of red light. So if we stimulate E. coli with different wavelengths of light we can activate different biochemical pathways. This is the long term goal of the project. We hope to control two competing biochemical pathways with little lag time using a reversible switch.

The bacteriophytochrome vector will be transformed into E. coli competent BL21 (DE3) cells, which contain 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 of DNA polymer synthesis, devised by DG Gibson et al. in 2009 (Gibson et al., 2009). The technique comprises an isothermal, single-reaction experiment involving the concerted action of a 5’-exonuclease, a DNA polymerase and a DNA ligase. An overview of the Gibson assembly method is provided in Figure 1 below and involves the ligation of DNA oligos, or ‘gBlocks’, of varying size (upto 500 BP each). As can be seen in the figure, gBlocks contain overlapping DNA regions of at least 30 BP which are exposed upon the action of the exonuclease. This facilitates complimentary base pairing between gBlocks. The simultaneous action of a DNA polymerase (preferably with proof-reading activity) and Taq polymerase, respectively, then complete and seal the newly formed conjugate (Gibson et al., 2010, Gibson et al., 2009).

The technique has obvious advantages in terms of time efficiency and simplicity. Indeed it involves less labour and steps to complete compared to traditional methods. In particular no polymerase cycling assembly; PCR, gel purification; restriction digestion and DNA ligation steps are necessary (Gibson et al., 2010). For these reasons we have chosen to apply this technique to the assembly our BioBricks.

Figure 1: An overview of the key steps in the Gibson assembly method for DNA polymer synthesis. (Figure adapted from DG Gibson et al.)