Our Project
A problem that arises in biosynthesis is that the overexpression of valuable metabolites can sometimes lead to cell toxicity. We propose the use of an optogenetic device to ensure that an essential late-stage enzyme involved in the production of a potentially toxic metabolite is expressed only at the optimal time.
The A. thaliana photoreceptor CRY2 and its binding partner CIB1 offer a rapid, reversible system for the control of cellular activity, through the fusion of split effector proteins that are reconstituted upon illumination with blue light. Due to the origins of optogenetics in neural cells and the high value of potential applications in human cells, the CRY2/CIB1 device has been widely applied in eukaryotic cells. Therefore we aimed to express the CRY2/CIB1 system in E. coli to show that this system also has diverse and useful applications in prokaryotic systems.
We hoped to use the CRY2/CIB1 system to regulate a split Cre-recombinase to induce the expression of a late-stage enzyme in the NAD biosynthesis pathway, thus maximising the yield of this valuable metabolite.
Experimental Work
In our experiments, we fused CRY2 and CIB1 to CFP and YFP (respectively) to see if the CRY2/CIB1 system was expressed and functional in E. coli by FRET analysis. Despite redesigning our FRET constructs to facilitate purification and functional testing and using different methods to construct our devices, while YFP was strongly expressed, CFP-CRY2 was poorly expressed in cell lysates.
Our results gathered over the course of the experiment increasingly suggested that fragments of the optogenetic device were too large for expression within E. coli. SDS PAGE analysis of CRY2-CreN and CFP-CRY2 pointed to a large proportion of insoluble CRY2 protein. Further, CIB1 fragments showed significant proteolysis, suggesting that despite the strong YFP expression seen in the FRET tests, the CIB1 fusion module may not be intact and able to associate with CRY2.
Overall, while we succeeded in creating and expressing our CRY2/CIB1 reporter FRET and CRY2/CIB1-Cre-recombinase constructs, insolubility of CRY2 and proteolysis of CIB1 suggests that they would not be able to associate upon blue-light induction to form a functional system.
However it was possible to record FRET tests using fluorescent proteins fused to CRY2 and CIB1 despite a large fraction of CRY2 being insoluble. These tests suggested that an energy transfer event occurred when compared to background dilution effects, which may involve the FAD chromophore of CRY2.
Assaying the functionality of the CRY2/CIB1-Cre-recombinase system presented another roadblock: even without any induction, our reporter construct (loxP-flanked RFP followed by a kanamycin-resistance gene) showed leaky read through, which was not reduced by the addition of a promoter after the first gene.
Proposed Designs
We aim to express CRY2-CreN and CIB1-CreC. Under dark conditions these constructs do not associate, but blue light allows the reversible binding of CRY2 to CIB1. Association of Cre-Recombinase halves (CreN and CrecC) in blue light conditions allows functional Cre-recombinase to excise targeted DNA between parallel, 34bp loxP sequences.
If the FRET and Cre-recombinase reporter constructs were functional, we hoped to apply this system to the biosynthesis of NAD, which is produced in E. coli either de novo or through salvage pathways regulated by the cell. We proposed that limiting the expression of a late-stage enzyme (nadA, quinolinate synthase) necessary for de novo NAD biosynthesis could potentially allow an accumulation of the precursor metabolite, iminoaspartate. Expression of nadA could then be induced by a blue-light activated Cre-recombinase.
By taking advantage of the accumulated intermediate metabolite, nadA could rapidly overexpress quinolinic acid, the subsequent metabolite in the production for NAD, before these high concentrations of NAD decrease cell fitness. In this way, the concentrations from biosynthesis of NAD could be greatly enhanced - an analogous method of biosynthesis has been used in the production of astaxanthin in microalgae.
Furthermore, in considering the scale-up of this potential system in an industrial setting, we researched the design of photobioreactors used for H2 production as well as an innovative biocontainment strategy using genetically recoded organisms dependent on synthetic amino acids.
Outreach
While the biosynthesis of valuable metabolites is becoming evermore common, genetic engineering within the public is increasingly regarded as exceedingly complex or inherently unethical. Therefore we engaged the broader community with our work and synthetic biology in general through outreach programs. We ran stalls as part of the National Science Week 2015 programme and held sessions at a local high school. We used simple analogies and visual demonstrations to explain concepts of genetics, protein function and optogenetics and facilitated discussion about the ethical issues surrounding synthetic biology in high school classrooms.
A Blue-Light Source
Further exploring the application of light-activated biotechnologies, we found that while a practical advantage of light is that it is cheap and readily accessible, previous research on CRY2 has used highly specialised and expensive blue-light sources. Therefore we have constructed and provided instructions for a cheap, simple and customisable blue-light source that can be made from readily accessible materials - and requires minimal experience with electric circuits to make! In particular our light source was able to vary intensity and pulse duration. We hope the inclusion of our device's construction on this wiki website will aid and encourage future teams to research optogenetic devices.
iGEM at ANU
Through taking part in the iGEM this year, as the first ANU team, we encountered a steep-learning curve in how to manage and carry out a project so widely-encompassing and demanding of time, creativity, skill, effort and teamwork. We hope that the evaluation of our experience from this year and the approaches we learn and connections we make at the Giant Jamboree facilitate only bigger and brighter directions for future ANU teams!
