Difference between revisions of "Team:ANU-Canberra/background"

Optogenetics is defined by the control of cellular dynamics using light. It allows for the ability to control the translocation of proteins, transcriptional activation and other potential uses. In its early stages, optogenetics focused on light-gated ion channels to control neuron excitability, but it is now expanding to broader applications.

Biosynthesis of valuable metabolites such as NAD and astaxanthin are areas of considerable research. A considerable stumbling block towards overexpression of these metabolites for commercial purposes arises when high concentrations of these compounds can become toxic towards cells and reduce cell fitness. One such strategy to mitigate these issues is to restrict the moment of over-expression of the compound until production is most efficient. We can use optogenetic tools to create an irreversible switch allows for the expression of an end-stage enzyme necessary for the production of the final metabolite.

In this project we aim to test for the functionality of a known eukaryotic optogenetic system, CRY2/CIB1 in prokaryotic E. coli. We will further test this system in conjunction with a split-Cre-recombinase construct that will enable the excision of targeted DNA constructs between parallel loxP sites only in the presence of blue light. Such a system could be used to control the expression of an end-stage enzyme required for the expression of a toxic metabolite such as NAD.

Light-based induction offers a more effective option to activate specific cellular activities than traditional chemical induction:

CRY2 (cryptochrome 2), is a photosensitive protein found in all kingdoms of life. Human cryptochrome 2 helps modulate circadian rhythm; in plants CRY2 regulates the timing of flowering. CRY2, isolated from Arabidopsis thaliana genome, has been demonstrated to reversibly bind to CIB1, a calcium binding protein of unknown function from the same organism, when irradiated with blue light (450nm wavelength). The basic strategy of an inducible, reversible CRY2/CIB1 optogenetic system is to bind an inactive, split effector protein of interest to form a functional construct. Irradiation of the light-inducible protein-interaction module with blue light brings two inactive protein fragments bound to CRY2 and CIB1 together consequently forming an active heterodimer.

CRY2 derived from A. thaliana (in which it initiates flowering) has been well-studied for the regulation of transcription; a further advantage of this photoreceptor is that it uses a chromophore endogenous to eukaryotes, flavin adenine dinucleotide (FAD).

The photosensitive protein CRY2 is stimulated by blue light (450nm) to bind to CIB1. Association of these proteins may be used to translocate proteins (A, B) fused to these constructs. In the absence of blue light CRY2 and CIB1 do not form a heterodimer, and the co-association of the two constructs is easily reversed in the absence of blue light.

Blue-light illumination activates CRY2 within seconds via reduction of the FAD chromophore, causing a conformational change that allows it to bind its interacting partner, cryptochrome-interacting basic helix-loop-helix1 (CIB1). After return to the dark, the CRY2/CIB1 complex dissociates in a matter of minutes.

This versatile strategy has been used to regulate transcriptional activation by split transcription factors, protein clustering to regulate cell signalling via oligomerisation of CRY2, and protein translocation through fusion of CIB1 with a form of eGFP which localises to the plasma membrane.

CRY2 systems have been primarily tested in eukaryotic systems, such as yeast, zebrafish and chinese hamster ovary cells. This is in part due to the origins of optogenetics in neural cells and the high value of potential applications in human cells. However, we aimed to express the CRY2/CIB1 system in E. coli. to show that this system has diverse and valuable applications in prokaryotic systems as well.

Cre-recombinase is an enzyme derived from P1 bacteriophage that is utilised in site-specific recombinase technology in both prokaryotes and eukaryotes. Cre-recombinase targets DNA recognition sites termed 34bp DNA loxP sequences. DNA between two parallel loxP is excised by active Cre-recombinase, whilst loxP sites inversely orientated inverts the intervening DNA. This enables the removal of specific genes when Cre-recombinase is active.

Temporal control of Cre-recombinase can be manipulated by splitting the protein at specific sites into two moieties (CRE-N, CRE-C) such that either moiety is inactive whilst forming a functional protein by ligand-induced dimerization. Past papers have used light-induced fusion of CRY2-Cre-N and CIB1-Cre-C constructs to form functional Cre-recombinase. The ability to initiate the excision of specific DNA sites with light enables the control over the expression of genes using light.

We hope to test for the activity of a reliable, non-reversible, blue light activated switch in E. coli. using split Cre-recombinase CRY2/CIB1 construct. This could potentially be used induce the production of alternative genes coding enzymatic pathways that contain a final component that is otherwise toxic to E. coli growth.

Overexpression of certain organic molecules or proteins, due to genetic engineering, can result in toxic levels of intermediate or end products resulting in decreased cell fitness. In particular, we are focusing on nadB and nadA bacterial genes (aspartate oxidase and quinolinate synthase respectively), that encode for enzymes required during Nicotinamide adenine dinucleotide de novo synthesis.

Nicotinamide adenine dinucleotide (NAD) can be produced via a six-step de novo biosynthetic pathway in bacteria. However the accumulation of high levels of NAD can be toxic to bacterial cells thus limiting the concentrations of NAD that can be achieved in bacteria for biomanufacturing purposes. We propose that suspending de novo production of NAD at a precursor stage until the activation of the CRY2/CIB1 Cre-recombinase complex induces the production of a subsequent enzyme necessary for the production of NAD. This could allow higher concentrations of NAD to be achieved in transformed cells before death due to toxicity.

CRY2/Cre-N and CIB1-CreC constructs do not form active Cre-recombinase in the dark. In this environment, the gene nadB can be expressed, whilst nadA is not due to the presence of a terminator upstream of the gene. Upon blue light stimulation, co-association of CRY2/CIB1 domains allows a functional Cre-recombinase enzyme to form. This enables the excision of nadB and the transcription of nadA.

This concept has not been trialled using CRY2/CIB1 device before. However an analogous strategy is the commercial production of astaxanthin, a carotenoid in high demand globally for use as pigments in feedstock and as a potential anti-cancer and immunostimulatory compound. Production in macroalgae provides an economical alternative to chemical synthesis and a two stage process maximises yield.

This resembles our strategy in the accumulation of a precursor (in this case, biomass capable of synthesising the product) and the subsequent induction of high-yield synthesis of the desired product (necessary in this case as high levels are only produced under certain conditions).