Due to the limitations created by COVID19. Our project has been remote this year, with zero lab access. But keeping in mind the challenges we faced during the design and implementation stages of our project. We decided to focus on providing teams with literature that is not only useful but enables them to improve their project in a practical manner. Our data seeks to improve the iGEM projects in the lab and their real-world implementation. Keeping this in mind, we have tackled problems that we ourselves faced during the workings of our project.
New Information for existing Parts
Tobacco Etch Virus nuclear-inclusion-a endopeptidase (TEV protease) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.
How are we using it?
We are using it to separate our effector transcription factors (PDX1, MAFA, and NGN3) in the cytoplasm of the intestinal crypt cells stoichiometrically.
TEV protease can be delivered with the genes of interest as a fusion protein to construct multi-gene circuits with a single vector. Using the TEV protease to cleave fusion proteins allows us to couple the translational rates of different factors to each other, enabling relatively stricter stoichiometric control over our systems. It has been used to efficiently and stoichiometrically separate up to three proteins in bacterial cells (E. Coli) as well as mammalian cells (COS-7, HeLa, and HEK-293)3. We codon-optimized the existing sequence for expression in Escherichia coli. Additionally, we removed the extra base pairs from the existing sequence and added a start codon for independent translation. (New part: BBa_K3450016)
Reference 3. Chen X, Pham E, Truong K. TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector. Protein Sci. 2010;19(12):2379-2388. doi:10.1002/pro.518
Microtubule Associated Proteins
Map20 is a signal sequence which encodes for a signal peptide which is used as an N-terminal tag for a protein to be translocated by the Escherichia coli type-3-secretion system (T3SS). This 20 amino acid signal peptide derived from the map gene is sufficient to direct proteins to the type-3-secretion system (T3SS) pathway.
How are we using it?
We are using the Map20 signal peptide to target our polyprotein to the T3SS pathway.
Map20 is the signal sequence of Map protein, encoded in a chromosomal pathogenicity island called locus for enterocyte effacement (LEE) and mediates secretion and translocation in a type III-dependent but chaperone-independent manner.1
Reference 1. Charpentier X, Oswald E. Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J Bacteriol. 2004;186(16):5486-5495. doi:10.1128/JB.186.16.5486-5495.2004
Glucagon-like peptide-1 (GLP-1) is a peptide hormone and is derived from proglucagon. It is an incretin molecule therefore, it can decrease the blood sugar levels in a glucose-dependent manner by stimulating the secretion of insulin.
GLP-1 has a half life of about 1-2 minutes in humans as it is actively degraded by dipeptidyl peptidase 4 (DPP4). DPP-4 resistant GLP-1 analogues and DPP-4 inhibitors are used as therapeutic agents for type-2 diabetes management.
Structure of GLP-1(1-37)
Reference Holst JJ. From the Incretin Concept and the Discovery of GLP-1 to Today's Diabetes Therapy. Front Endocrinol (Lausanne). 2019;10:260. Published 2019 Apr 26. doi:10.3389/fendo.2019.00260
Exendin-4 is a glucagon-like peptide 1 (GLP-1) receptor agonist and was discovered in the venom of Heloderma suspectum. Exendin-4 has a considerably higher intravenous half-life in humans (26 minutes) than GLP-1 and can be used for the treatment of type-2 diabetes mellitus.1
Recent reports revealed its role in suppression of PI3K/Akt/mTOR pathway and inhibition of enzalutamide induced invasion and migration of prostate cancer cells.2 Exendin-4 and enzalutamide treatment might be beneficial for patients with advanced prostate cancer3. Additionally, we added the stop codon and codon-optimized the existing sequence for expression in Escherichia coli (New part: BBa_K3450020).
Simonsen, L., Holst, J.J. & Deacon, C.F. Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetised pigs. Diabetologia 49, 706–712 (2006).
He W, Li J. Exendin-4 enhances radiation response of prostate cancer. The Prostate. 2018;1–9.
He W, Shao Y, Yu Y, Huang W, Feng G, Li J. Exendin‐4 enhances the sensitivity of prostate cancer to enzalutamide by targeting Akt activation. The Prostate. 2020;1–9.
CesT is a chaperone that facilitates secretion of effector proteins like Map and Tir by E. Coli Type-3 secretion system (T3SS).1
Recent studies have reported its role in reprogramming of gene expression in bacteria attached to its host cell via inhibition of a post transcriptional regulator Carbon storage regulator A (CsrA)3. Translocation of a major effector protein Tir is dependent on CesT. CesT is liberated after translocation of Tir protein2,3. The liberated CesT reprograms the gene expression in host-attached bacteria as it binds to and inhibits the post transcriptional regulator CsrA3. Additionally, we codon-optimized the existing sequence for expression in Escherichia coli (New part: BBa_K3450019).
Structure of CesT (dimer)
Thomas, N. A. et al. CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli. 57, 1762–1779 (2005).
Ye, F., Yang, F., Yu, R. et al. Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT. Nat Commun 9, 1196 (2018).
Elbaz N, Socol Y, Katsowich N, Rosenshine I. 2019. Control of type III secretion system effector/chaperone ratio fosters pathogen adaptation to host-adherent lifestyle. mBio 10:e02074-19
THE BIG PIE is a project aimed to provide an alternate therapy for insulin-dependent Diabetes Patients. Our pill iBeta is relevant to the healthcare and pharmaceutical industries. Considering the reach of our product, we decided to find out the patenting process of our pill. However, iGEM mainly contained patent guidelines for American and European teams. Drawing inspiration from these teams, we spoke with Indian patent officers to help us understand the patenting procedure for our pill in our country. Our interactions with patent officers and information from the drug development guidelines have made us aware that our pill iBeta is a novel therapeutic using lab modified bacteria and hence would be classified as a “New Drug”. Keeping in mind the policies and regulations for lab modified bacteria and synthetic biology in India, we decided to create a patent guide for all future Indian iGEM teams that would be interested in taking their project beyond the lab. We hope this helps you all!
Additionally, we optimized and removed the extra bases from the coding sequences of the following biobricks for expression in Escherichia coli. (old part, new part) (BBa_K2871004,BBa_K3450019) (BBa_K3096030,BBa_K3450020) (BBa_K1639008, BBa_K3450016)