Escherichia coli is a major chassis used in the biotechnology industry to produce recombinant therapeutic proteins. To quantify it, nearly 30% of the industry works upon E. coli for expression of non-glycosylated proteins. Major reasons for this are its well characterised genetics, rapid growth and high yield production. Today more that 151 unique recombinant therapeutics made by E. coli have been approved by the FDA for different clinical purposes.
Even after years of work and advances, the major issue faced by industry still lies in the part of protein translation. Protein translation has four major phases: initiation, elongation, termination and ribosome cycling. In most cases, translation initiation is a rate limiting step for protein biosynthesis. Hence with our iGEM project, we decided to focus on finding ways to optimise translation initiation which is the burning need of the industry. Translation initiation efficiency is determined by the sequence and structure of the translation initiation region (TIR) consists of four major sequences, the Shine Dalgarno sequence, the start codon, spacer region, and translational enhancers called 5' UTR located upstream of the SD sequence. We have focused our project mainly towards these UTRs as this domain has been relatively less looked at, and has a great scope for advancement. We are trying to establish a library of standardised 5' UTRs with laboratory-determined expression rates for various proteins, which can be used directly to optimise yields in a laboratory or industrial set-up.
We have found that even small mutations as low as 1 base pair can vary the expressions greatly. With our experiments and dry-lab computations, we have determined 5' UTR sequences with expression rates as low as 30% and as high as 200% of the original, native 5' UTRs. We feel using modified recombinant E. coli can lead to significantly higher yields and hence impact industrial production of therapeutic drugs greatly thereby reducing costs of drugs amongst other benefits.
Our implementation plan involves developing a library for the Dh5α and BL21(DE3) strains of E. coli. We also aim at testing it out in the MG1655 strain of the same organism, and making modifications based on our experimentally validated data, the neural network developed by us as well as the data generated by pre-existing software tools. We wish to work on conditional expression as well and modify our UTRs accordingly as suggested by Amanda Hughes. This would be a constant iterative process, supplemented by feedback from industrialists and researchers. We would simultaneously be re-evaluating safety features of our organisms if the need arises, look into intellectual property rights to protect our findings, and seek out funding options to increase our scope of operations. Our basic library of parts would be open-source.
As implicated by our literature search, the region downstream of the coding sequence does play a role in the effect of translation initiation but eventually we would be adding a cleavage site there in our basic library. Then, we would also be involving ourselves in projects with industries and providing them with a special custom library for their gene of interest which would be maintained as a trade secret (In this case no changes to the CDS would be made). We would be performing scale up experiments on both a laboratory level and an industrial level. There is a concern on the performance of the cells and UTRs once the volume of the reactor changes. We would be recording these differences, making attempts to understand them better, and ideally make our optimization strategies further enhanced. In cases where a cleavage site is added to standardise the 5' UTRs, characterisation of the protein would be required again.
Our PI has done a diploma in Patents Law and is also a part of the Safety Committee of our Institute. We are constantly in check with respect to our safety aspects. We have stated everything in the safety form. Our Meets with Mr. Soham D'Souza and Mr. Nilesh Kulkarni gave us better industrial insights.
Dr. Narendra Chirmule suggested that we should also work on protecting our Intellectual Property Rights. The sequences that we would be making available open source would be available for everyone and the future iGEM Teams. However if we specifically optimise the sequence for a particular industrial project, that would be maintained confidential. He also cautioned us that it might be difficult for us to incorporate this process in already established protocols. The companies may need to work on the downstream processing again. We should instead be looking at novel proteins or biotechnology based startups. This gave us further thoughts. Rare disease proteins like Factor VIII are not manufactured in our country. It would be beneficial to implement our process in such low volume processing. And taking a step further in the thought process from a business perspective, the relatives of the members of person suffering from a rare disease are usually more than willing to contribute and financially support research and development in that field.
Further, in our meeting with Mr. Manish Mishra who handles the India business and South Asia Venture Capitalist practice for SBI Holdings (Softbank Investment Holdings INC JP), particularly looking for innovations in green chemistry and biotechnology, we realised that through our process if we are optimizing the yields we may also be saving carbon credits. We intend to look at how carbon credits are calculated for biotechnological processes and how we could create a greener and greater impact. We further understood more about Respect, Diversity, Honesty, Responsiveness, Cooperation, Safety from the Values and Risks Workshop of iGEM. EPIC explained Product Market, Business Modelling and Pitching. The learnings from this are being consciously incorporated in our project.
Values and Risks Workshop
