Team:Macquarie Australia/Project

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Aim

The main objective of our project is to introduce Deinococcus radiodurans and Agrobacterium tumefaciens bacteriophytochromes into E. coli which have the potential to be used as molecular light switches in response to red and far-red light. Comparison and analysis of the phosphorylated peptides in recombinant E. coli can also be considered in the future. The following are our four main aims which we strive to achieve by iGEM in early November 2010.

How we plan to achieve this:

1. Isolate a bacteriophytochrome gene: from two bacterial species Agrobacterium tumefaciens and Deinococcus radiodurans.

2. Clone: the bacteriophytochrome into a pET-3A vector that contains the heme oxygenase gene and transform it into an E. coli strain, BL21(DE3). Primers will be designed specifically so that the bacteriophytochrome gene is amplified with both a ribosome binding site and a heme oxygenase site.

3. Create a biological light switching mechanism. We expect that the E. coli transformants will express both Biliverdin produced by the heme oxygenase enzyme and the bacteriophytochrome protein enabling the E. coli to turn from blue to green when red and far-red light is absorbed by the colonies. This particular light switching mechanism has not been obtained in E. coli before. This aim is therefore particularly significant in that a novel function is being genetically synthesized in a model organism allowing further research into the effects of genetically engineering this switching effect in E. coli.

Abstract

Photoreceptors are utilized by almost every organism to adapt to their ambient light environment.

Our aim is to engineer a novel reversible molecular ‘light switch’ within E. coli by introducing a photoreceptor from non-photosynthetic bacteria ( D. radiodurans and A. tumafaciens ).

By cloning the bacteriophytochorome coupled with heme-oxygenase, an enzyme that produces biliverdin from heme, the created colonies are able to respond to red and far-red light environmments.

This novel approach results in the colour of the E. coli ‘switching’ from blue to green.

Our E. coli chameleon will serve as a fundamental ‘bio-brick’ for future applications by providing a simple and photo-reversible switch.

Background

Phytochromes are red/far red light sensors which use the phytochromobilin chromophore to act as photoreceptors which can regulate, in photosynthetic organisms, the growth, germination and other factors effected by light (Essen et al, 2008).

Phytochromes are proteins found in higher plants and photosynthetic bacteria. Research into the field of phytochromes, outside of the various autotrophic biological systems including plants and other photosynthetic organisms, has recently discovered that a form of phytochome exists within heterotrophic bacteria including Agrobacterium tumefaciens and Deinococcus radiodurans (Bhoo et al, 2008).

Further research has also found similar protein families in fungi and cyanobacteria (Karniol et al, 2001). The family of proteins found in bacteria are similar to phytochromes having phytochrome-like sensor kinases to enable bacteria to adapt to different forms of light in their surroundings. This family of proteins in bacteria are known as bacteriophytochrome photoreceptors (BphPs) (Karniol et al, 2003; Vierstra et al, 2000).

The biliverdin chromophore, a greenish bile pigment, is a product of the breakdown of heme by heme oxygenase (HO). This reaction, driven by the inducible enzyme HO, catalyses the first and rate-limiting step in the oxidative degradation of free heme into ferrous iron, carbon monoxide, and biliverdin (Rockwell et al, 2006).

For the light response by phytochromes to take place, the phytochrome must first bind a chromophore which is typically a linear tetrapyrrolic molecule (or billin). The phytochrome-billin complex can undergo photointerconversion between two stable isoforms which allows proteins to respond to changes in light in the surrounding environment (Bhoo et al, 2001). In the absence of light, the complex forms a red light absorbing isoforms (Pr).

When this form is excited with red light, it undergoes a conformational change to form the far-red light absorbing isoforms (Pfr) whose activity is repressed by far-red light (Davis et al, 2007). Because of this reversible interconversion, they have been referred to as photo-reversible molecular switches. Many phytochromes include a photosensory cascade domain that allows downstream signal transduction processes to take place via a series of phosphorylation events (Vuillet et al, 2007).

An external light signal is transduced into an internal chemical signal allowing the plant to adapt to its outside environment by prompting a diverse range of growth and developmental responses which include seed germination and shade avoidance strategies (Yang et al, 2008; Wagner et al, 2007). Through genome wide searches using known phytochrome sequences, these proteins have also been identified in species of fungi and bacteria. BphPs have been identified in various non-photosynthetic bacterial species such as Deinococcus radiodurans, Pseudomonas aeruginosa, and Agrobacterium tumefaciens and their action has been implicated in a variety of sensory responses including chemotaxis and pigmentation (Lamparter et al, 2003).

Biliverdin covalently attaches to the chromophore binding pocket in the bacteriophytochrome. These chromophore binding sites are the structurally conserved regions identified in BphPs (Quail, 2010). This photosensory cascade, is found at the C-termini, causes the conformational change and essentially creates a photoreversible switch mechanism within the organism, which mediates the conformational change enabling the molecule to exhibit different colous- blue and green(Rockwell et al, 2006).

Expected outcomes of our project