Team:St Andrews/Design
Design
Motivation
As residents of a sunny seaside village, we were inspired to help preserve the local marine environment without discouraging tourists from enjoying its beauty. Research confirmed the need for a sustainable sunscreen, and the inadequacy of existing products.
Using the aromatic compound shinorine, already native to marine species such as red algae and cyanobacteria, the molecule can effectively be used as a natural UV-absorbing component of sunscreen, where production has been previously achieved by various research groups (Balskus and Walsh, 2010; Miyamoto et al., 2014). Unlike toxic sunscreen ingredients, shinorine would efficiently absorb UV radiation and possess no environmental threat to aquatic systems.
A recent emphasis on improving translational efficiency of the shinorine-producing enzymes in model organisms such as E.coli has been shown (Yang et al., 2018) . The Minnesota iGEM team of 2012 looked to improve upon the efficiency of UV protection using a similar system to that of Balskus and Walsh (2010).
Their goal was to clone multiple gene pathways responsible to produce shinorine alongside other UV-absorbing compounds. The team were able to successfully transform and produce shinorine and its precursor mycosporine-glycine despite expression issues with the final gene of the synthesis pathway. Yang et al. (2018) highlighted an additional issue with the final step as the low catalytic efficiency of the enzyme limited the rate of shinorine production.
Our team also carried out research into widely available commercial sunscreen ingredients. We found many ingredients such as oxybenzone and octinoxate to be present in most common water sources. Oxybenzone has been determined to contribute to coral bleaching effects whilst other UV filters have been seen to bioaccumulate within the food chain (Schneider and Lim, 2018). To protect aquatic ecosystems such as coral reef systems, Hawaii became the first U.S. state to pass a bill prohibiting the sale and distribution of sunscreens containing the above compounds. With the U.S. Virgin Islands and Palau recently following suit, it is evident that a sustainable, non-toxic alternative to sunscreen is desirable.
From research, the idea of producing synthetic free-radical scavengers in response to UVA intensity was suggested. This prospect quickly associated itself with shinorine as our feature UV-absorbing compound. With the evolution of next generation sequencing and metagenomics, we also began to consider, rather than isolating shinorine from culture before applying to the skin, could we apply the bacteria directly to produce the compound in vivo? The bacteria would then remain on the skin accumulating shinorine within the cell to provide a layer of UV protection before inactivation or strain termination was initiated by external factors.
Design
Shinorine Producing Cluster
From the variety of organisms possessing the shinorine gene cluster (Miyamoto et al., 2014), production of shinorine by transformation into E.coli from A.variabilis was shown previously to be successful (Balskus and Walsh, 2010). The gene cassette for the pathway involves Ava_3858, Ava_3857,Ava_3856 and Ava_3855 coding for DHQS, O-MT, ATP-Grasp and NRPS-like enzymes respectively. Gene sequences were subject to the IDT codon optimisation tool to improve transcriptional efficiency in our E.coli chassis (sequences can be found in parts BBa_K3634000, BBa_K3634001, BBa_K3634002 and BBa_K3634003).
The four gene cluster was split into two separate operons, the first coding for DHQS and O-MT and the second ATPG and NRPS. Through literature and modelling data, it was found that both ATPG and NRPS enzymes were rate-limiting in the pathway (Yang et al., 2018). As a result, both genes were given independent constitutive promoters and RBS, shown in BBa_K3634004. Both DHQS and O-MT were under the control of only one constitutive promoter, shown in BBa_K3634004.
We further considered various pathways generating intermediates essential for the shinorine synthesis pathway such as those from the shikimate and pentose phosphate pathway (PPP) in order to optimise shinorine production. The transaldolase enzyme (tal) involved in the PPP converts glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate (SH7P) to fructose 6-phosphate and erythrose 4-phosphate. As SH7P is the initial substrate required in the shinorine pathway, knockout of tal would allow the substrate to be accumulated. Furthermore, over-expression of gnd encoding 6-phosphogluconate dehydrogenase by promoter exchange was found to increase shinorine production by 60% (Tsuge et al., 2014). This modification was also taken into consideration.
Figure 1. Shinorine Enzymes. Basic parts BBa_K3634000-BBa_K3634003 drawn respective to gene length. BBa_K3634004 and BBa_K3634005 composite parts also shown. Promoter (Light Green); RBS (Dark Green); Terminator (Pink).
Kill Switch Component
Developing a probiotic sunscreen immediately presented both biosafety and biosecurity risks including escape of our probiotic into the surrounding environment (the environment being anywhere except the skin surface) and horizontal gene transfer (HGT) of our synthetic constructs to other skin microbes. After careful consideration and multiple versions, an intricate kill switch mechanism was devised, implementing a novel two plasmid dependency design and complex sensing system to terminate bacterial survival when UV protection is no longer required.
Initial research into the kill switch component focused on toxin antitoxin modules (TA), temperature-dependence, memory elements and evolutionary stability (Wright et al., 2013; Chan et al., 2016; Stirling et al., 2017). Our design thereafter focused primarily on sensing systems to moderate death of our bacteria through regulation of a TA system.
Both temperature and glucose regulatory kill switches were suggested but after discussion surrounding the source of the temperature sensor (from Salmonella typhimurium), this aspect was archived on biosafety terms. As a replacement, we had previously been using a UV-sensing system UirS/UirR (Ramakrishnan and Tabor, 2016) for transcriptional output of the shinorine gene cluster. After discussion with Dr. Helder Ferreira of the University of St. Andrews, we chose to maximise expression of shinorine production by putting the cluster under constitutive control, instead using the UiS/UirR system for the kill switch. The discussion with Dr. Helder Ferreira prompted further investigation into the UV-sensing system itself where it was later decided that light could instead be used as a general indicator of UV allowing for the system to be replaced with a more robust, efficient light sensor (CcaS/CcaR) (Tabor et al., 2011).
Figure 2. Two Component Light Sensing System. Basic parts BBa_K3634006, BBa_K3634007, BBa_K3634008, BBa_K3634017 drawn respective to gene length. Composite parts BBa_K3634009 and BBa_K3634019 also drawn. Promoter (Light Green); RBS (Dark Green); Terminator (Pink).
As for the TA system we planned to use, an initial sib sRNA strategy was proposed as already native to E.coli and also able to modify proteins activity or base pair with mRNA (Fozo et al., 2008). This strategy was abandoned however due to the complexity of the system and the unknown mechanism of toxicity. Additional TA module systems were then investigated, preferably with the ability to destroy the cell and plasmid content. The ccdAB TA system was selected as it remains the best characterised toxin antitoxin module available and is native to E.coli so no codon optimisation was required. As the system mostly promotes bacterial stasis and not DNA degradation, a second endonuclease (R.CviJI) was integrated to shred the plasmid constructs preventing HGT.
Figure 3. Glucose-Mediated Death Sensor (BBa_K3634012), CcdAB-Mediated mf-Lon Protease (BBa_K3634014) and Tagged LacI Repressor (BBa_K3634016). Associated basic parts BBa_K3634011, BBa_K3634013 and BBa_K3634015 are shown respectively. Promoter (Light Green); RBS (Dark Green); Terminator (Pink); Degradation Tag (Red).
Figure 4. LacI-Controlled CviJI Endonuclease (+ssRA Degradation Tag) (BBa_K3634021). Promoter (Light Green); RBS (Dark Green); Terminator (Pink); Degradation Tag (Black).
As for the specific strain of E.coli, Nissle 1917 was chosen as this organism is commonly used in other probiotics for the human gut and is crucially non-pathogenic and FDA approved. Based off this information alongside the fact that Enterobacteriaceae do not commonly populate the skin surface, after a certain duration, it was hypothesized that all initial E.coli and daughter cells would be outcompeted by the native skin flora.
References
Balskus E.P., Walsh C.T. 2010. The Genetic and Molecular Basis for Sunscreen Biosynthesis in Cyanobacteria. Science. 329(5999): p1653-1656.
Miyamoto K.T., Komatsu M., Ikeda H. 2014. Discovery of gene cluster from mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression. Appl Environ Microbiol. 80(16): p5028-5036.
Yang G., Cozad M.A., Holland D.A., Zhang Y., Leusch H., Ding Y. 2018. Photosynthetic Production of Sunscreen Shinorine Using an Engineered Cyanobacterium. ACS Synth. Biol. 7: p664-671.
Schneider S.L., Lim H.W. 2018. Review of environmental effects of oxybenzone and other sunscreen active ingredients. J Am Acad Dermatol. 80(1): p266-271.
Tsuge Y., Kawaguchi H., Yamamoto S., Nishigami Y., Sota M., Ogino C., Kondo A. 2014. Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine. Bioscience, Biotechnology, and Biochemistry. 82(7): p1252-1259.
Wright O., Stan G-B., Ellis T. 2013. Building-in biosafety for synthetic biology. Microbiology. 159: p1221-1235.
Chan C.T.Y., Lee J.W., Cameron D.E., Bashor C.J., Collins J.J. 2016. “Deadman” and “Passcode” microbial kill switches for bacterial containment. Nat Chem. Biol. 12(2): p82-86.
Stirling F., Bitzan L., O’Keefe S., Redfield E., Oliver J.W.K., Way J., Silver P.A. 2017. Rational Design of Evolutionary Stable Microbial Kill Switches. Mol. Cell. 68: p686-697.
Ramakrishnan P., Tabor JJ. 2016. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E.coli. ACS Synth. Biol. 5: p733-740.
Tabor JJ., Levskaya A., Voigt CA. 2011. Multichromatic control of gene expression in Escherichia coli. J. Mol. Biol. 405(2): p315-324.
Fozo E. M., Kawano M., Fontaine F., Kaya Y., Mendieta K. S., Jones K. L., Ocampo A., Rudd K. E., Storz G. 2008. Repression of small toxic protein synthesis by the Sib and OhsC small RNAs. Molecular microbiology, 70(5): p1076–1093.