Team:ANU-Canberra/Description
Project Description
2015 International Year of Light
This year being The International Year of Light and Light-based Technologies, we were inspired to investigate potential uses of light in synthetic biology. In particular we were fascinated by the novel research in optogenetics that allows for the spatial and temporal control of cellular dynamics using light.
Light-Inducible Protein-Interaction Module
CRY2 is a photosensitive protein found in all kingdoms of life. In humans it helps modulate circadian rhythm, in plants it regulates timing of flowering. We used CRY2 protein from A. thaliana. CRY2 has been demonstrated to reversibly bind to CIB1 when irradiated with blue light (420nm wavelength). The basic strategy of an inducible CRY2/CIB1 system is to control a split protein of interest. Irradiation of the light-inducible protein-interaction module with blue light brings two inactive protein fragments fused to CRY2 and CIB1 together consequently forming an active heterodimer.
[[File:Team_ANU-Canberra_CRY2-CIB1.png]]Cre-Lox Recombination
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 LoxP sequences. DNA between two LoxP sites oriented in the same direction is excised by active Cre-recombinase, whilst LoxP sites inversely orientated inverts the intervening DNA.
Temporal control of Cre-recombinase can be exerted 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. Light-induced fusion of CRY2-Cre-N and CIB1-Cre-C constructs forms functional Cre-recombinase enabling recombination of DNA at target sites.
Proposed DNA Structures
To apply the CRY2/CIB1 system to Cre-lox recombination using two reporter proteins in E. coli; showing that the first reporter protein is expressed in the dark without excision and the second is expressed upon blue-light illumination with excision.
[[File:Team ANU-Canberra-DNA.png]]Potential Functions
We hope to develop a reliable, non-reversible, blue light activated switch for inducing the production of alternative genes coding enzymatic pathways that contain a final component that is otherwise toxic to E. coli growth. 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. Precursors of NAD can be important nutritional supplements in humans and animals. However the accumulation of high levels of NAD can be toxic to bacterial cells which thus limits the concentrations of NAD that can be achieved in bacteria for biomanufacturing purposes. We propose 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.
[[File:Team:ANU-Canberra/Team_ANU-Canberra-DNA.png]]References
Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D., Tucker, C.L. (2010) “Rapid blue-light-mediated induction of protein interactions in living cells” Nature Methods vol. 7 no. 12 pp. 973 - 975
Begley, T.P., Kinsland, C., Mehl, R.A., Osterman, A., Dorrestein, P. (2001) “The Biosynthesis of Nicotinamide Adenine Dinucleotides in Bacteria: Vitamins and Hormones, Vol. 61” Academic Press pp. 103 - 119