1. New application prospects of iGEM
From the first brainstorming session, we tried to think outside our comfort zone. This year is the first time that our HiZJU-China iGEM team has jumped out of the environmental track and opted for a therapeutic track that has never been tried before. We were very surprised to find that almost no projects in synthetic biology have medically used microbial contact lenses for the treatment of eye diseases (except NCKU-Tainan 2020). Therefore, we believe that our most important contribution to the future iGEM team is innovation in this field.
Through interviews with the Drug Administration, we learned that the current categories of medical products generally only have two categories of "drugs" and "devices", and our microbial contact lenses are a combination of "drugs" and "devices", which has few examples in safety supervision. But we believe that our microbial contact lenses will be a start, and in the future, products combining "drugs" and "devices" will surely create greater value with greater advantages.
In addition, we are inspired by NCKU-Tainan 2020 for embedding engineered bacteria in contact lenses, but unlike them, we have made great innovations in the improvement of biosafety. We added liposome vesicles to the inside of the contact lens, that is, a phospholipid membrane embedded with salidroside transporter protein as a shell, embedded in the bacterial solution, and encapsulated between the two layers of the contact lens membrane, as detailed in our Design page. In this way, the shipment of salidroside can be strictly controlled, while the bacterial solution and its metabolic waste will not be leaked.
Fig.1 Contact lens design(Fig a shows the storage conditions of biolens, Fig b shows the growth trigger conditions, and Fig c and d show the production trigger mechanism)
2. New parts on the registry
2.1 Fusion express
Fusion expression of enzymes corresponding to two adjacent reactions in the metabolic pathway may have an impact on the reaction rate. We designed and compared three flexible connections and three rigid connections, and found that connecting the adh6 and ARO10 genes with the flexible Linker3 connector significantly increased yields (see our Results page). Although we don't know if it will still work well when used to connect genes other than adh6 and ARO10, we are sharing the flexible connector Linker3 with future iGEM teams that may need it.
· Linker3 (BBa_K4761032): Used for adh6-ARO10 fusion expression.
Fig.2 The design of linker
2.2 Gene integration in E. coli
We designs to integrate ARO10 gene and UGT85A1 gene into E. coli using CRISPR RNA-guided integrases, we finally successfully verified the validity of our selected Escherichia coli gene integration sites. So we think this can help future iGEM teams.
· pTarget (pEffector) (BBa_K4761052): Expresses a guide RNA sequence to locate the desired sequence for gene knock-in.
· pEffector (BBa_K4761110): Expression box of the guide RNA.
· pDonor (BBa_K47611108): Amplifying exogenous gene fragments in the organism.
· pCutamp (BBa_K47611109): Containing genes such as Cas9.
Fig.3 Schematic of INTEGRATE using a Type I-F V. cholerae CRISPR-transposon[1]
3. Other experimental experience
When conducting molecular biology experiments, we are fortunate to complete most of the most critical copy number optimization, gene knockout, fusion expression, and gene integration experiments. However, the process of the experiment was extremely difficult. From mid-July to early October, we experienced many rounds of experimental failures and accumulated many lessons in the "Design-Build-Test-Learn" project cycle. Since these lessons may provide lessons for future teams, we present them on our Engineering Success page in order to avoid repeating them.