Shinescreen: A Novel,Entirely Reef-Safe Probiotic Sunscreen.

Biosafety

Biosafety is an integral part of every iGEM project, but it is particularly vital to projects such as our own that aim to develop a product that will be used by the general public and is therefore at higher risk of escaping into the environment.

Throughout our project, the biosafety has been a focus of ours. Due to the global pandemic and the UK lockdown, our project is entirely virtual. Therefore, considerations of lab practice are less relevant to our project, although we have considered how this may take place in the future and the precautions we will take. This year, our biosafety has been focused on minimising escape into the environment by designing a robust and evolutionarily stable gene circuit.

Our gene circuit is divided over two plasmids, with key genes spread between both. The shinogen component has four genes, with two on each of the plasmids:

pSB3B1-DOPH contains:
- 3-dehydroquinate synthase (DHQS) (BBa_K3634000)
- O-methyltransferase (O-MT) (BBa_K3634001)

pSB3E1-AN contains:
- ATP-Grasp (ATPG) (BBa_K3634002)
- non-ribosomal peptide synthetase (NRPS) (BBa_K3634003)

More information on our killswitch design can be found in Partsoverview.

The separation of the shinorine producing genes onto two plasmids minimises the risk of these genes escaping into the environment. In order to successfully produce shinorine, both plasmids are required. The chances of a member of the native skin flora successfully acquiring both plasmids are significantly lower than if all the genes were contained on a single plasmid.

The killswitch genes are also split between both plasmids:

pSB3B1-DOPH
- Light Sensor + ccdA Output (BBa_K3634019)
- R. CviJI Endonuclease (BBa_K3634021)

pSB3E1-AN
- Glucose Sensor + ccdB (BBa_K3634012)
- Mf-Lon Protease (BBa_K3634014)
- LacI + mf-Lon degradation tag (BBa_K3634016)

The genes are divided in such a way that ensures that the presence of one plasmid but not the other is deleterious to the bacteria, not only preventing escape of the genes, but also acting as a positive selection pressure for retaining both plasmids.

We also incorporated a degree of redundancy into the plasmid in the form of an R. CviJI endonuclease (BBa_K3634021), which significantly degrades the DNA of our system once activated. An in silico digest of plasmid B was performed, showing that the R. CviJI endonuclease digested the 4805bp plasmid into 75 fragments. These make the kill switch more evolutionarily robust – having a redundant system means that disabling mutations in one or more components does not necessarily mean that the kill switch itself is disabled. We hope that this makes our killswitch more evolutionary stable and decreases the likelihood of escape into the environment.

Ideally, we would demonstrate this in the lab, but due to our lack of access, we decided to instead simulate this using a modified version of the genetic algorithm. See our Modellingpage for more detail.

General Safety

Organisms Used

E.coli Nissle 1917 (Chassis)
DH5a E.coli (Cloning)

All these organisms are classified as non-pathogenic/non-toxic and are risk group 1 organisms. However, for all strains used, appropriate PPE including (but not limited to) laboratory coats, disposable gloves and safety glasses should be worn in 2021. Hands should be thoroughly washed with soap before and after any experimentation to avoid contamination/ingestion of organisms. If ingestion does occur, wash the mouth thoroughly and consider seeking further NHS guidance. If in contact with eyes, flush with cold water for 10-15 minutes. If skin irritation occurs, wash the area with soap and water. Both bacterial strains are RG-1 organisms and therefore possess minimal risk to the community. Without appropriate culture conditions, the bacterial strains will be rapidly outcompeted in the external environment.

Future Experimental Risks

The shinorine product is not a hazard to biological life. Good laboratory practice will be demonstrated when transforming the bacteria into the desired recombinant organism. Care will be taken when testing the UV absorption of our compound to ensure minimum human exposure to UV radiation.

Managing Further Risks

Training from the safety coordinator alongside an awareness for biosafety and biosecurity guidelines will help the team carry out safe biological transformations in the lab minimising the risk of infection to those individuals carrying out experimentation and release of the modified organism into the surrounding environment. Biobricks and our chassis were selected on the basis of reducing the pathogenic risk associated with our engineered organism. We aimed to make our project the least invasive as possible to not affect the natural distribution of bacteria present on the epidermis of the skin. Our chassis was selected as to reduce pathogenicity despite the availability of other organisms exhibiting surface lectins that may have allowed better attachment to the skin surface. The kill switch was designed as to limit bacterial survival only to the skin surface and prevent escape and further proliferation/HGT in non-permissive conditions/environments.

Safety, Security and Ethical Concerns

Although the chosen chassis is non-pathogenic and predicted to be rapidly outcompeted by the natural skin flora, there remains a possibility that our recombinant organism could become part of the host skin microbiome. E.coli are a common commensal of the gut microbiome so if transferred to this environment, conditions would be much more favourable for our engineered strain to populate. Both the glucose and light-dependent kill switch mechanisms would ensure the above scenarios would not take place. Horizontal gene transfer between species of the skin microbiome is a possibility and of those engineered genes, the shinorine cluster would most likely confer an advantage to skin microbiome species. Natural persisters of the toxin within the population may also result. As aforementioned, the two-plasmid system would act to minimise any advantage conferred to surrounding organisms. The AND/OR-gated endonuclease activation would also ensure fragmentation of the plasmid inserts following loss of permissive conditions, thus reducing the chance of functioning protein products in skin microbial species. Safety trials would be required before the proposed bacteria could be used as a probiotic sunscreen. Release of E.coli into aquatic systems may also be a potential risk however inclusion of the kill switch would minimise this. Further modelling has been carried out to determine the number of generations required before a mutation to the kill switch would occur inactivating this component.