Part:BBa_M50447:Design

Notes

The thinking behind this project was a therapeutic protein factory for insulin. People who have type 1 diabetes cannot naturally sense when blood glucose is high, and they are at risk for things like heart disease, stroke, kidney damage and nerve damage. We decided to synthesize this part in yeast because we know yeast can live in the gut microbiome, suggesting that our device could sense when glucose levels are high and secrete insulin in the digestive tract.

Nevertheless, we hypothesize that there is an intestinal mucus that would prevent the insulin from being reabsorbed into the bloodstream, ultimately rendering our part from being entirely practical. As such, we wanted this device to be a proof of concept device showing that the glucose-sensitive promoter works.

Source:

We got a lot of the DNA sequence from our TA for Bioe 44 in spring 2018, whose name is Matias. He advised us to synthesize this part in yeast, and he first told us to look for a T2A peptide. We got the T2A peptide from Addgene. The single chain insulin (which is considered to be ultra stable and resembles the native form of the protein) was sourced from Gidden et al., link: http://www.jbc.org/content/293/1/47. Other parts were sourced from previous iGEM teams and iGEM biobricks.

Importantly, we got our sequence for the promoter from iGEM14_Tshingua. They reported that their promoter expressed GFP when glucose concentrations were high and "turned off" when glucose levels were low, guiding our intuition to select this promoter that we wished would mimic pancreatic beta cells.

The composite part is long (about 2200 bp) and took a while to be synthesized and sequenced. It did not work as intended i.e. we did not see higher levels of GFP expression or higher concentrations of insulin protein when compared to negative controls.

References:

Diabetes: Facts, Statistics, and You. Healthline. https://www.healthline.com/health/diabetes/facts-statistics-infographic#1. Accessed May 9, 2018. The Cost of Diabetes. American Diabetes Association. http://www.diabetes.org/advocacy/news-events/cost-of-diabetes.html. Accessed May 9, 2018. Claesan J, Fischbach MA. Synthetic Microbes As Drug Delivery Systems. https://pubs.acs.org/doi/pdf/10.1021/sb500258b. Published 2015. Potera C. Bioengineering Bacteria for Drug Delivery. GEN. https://www.genengnews.com/gen-articles/bioengineering-bacteria-for-drug-delivery/2011. Published February 15, 2007. Accessed May 8, 2018. Hou L. Part:BBa_K1328002. Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_K1328002. Published October 9, 2014. Accessed May 9, 2018. Glidden MD, Aldabbagh K, Phillips NB, et al. An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein. J Biol Chem. 2018;293(1):47-68. Liu Z, Chen O, Wall JBJ, et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Advances in pediatrics. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438344/. Published May 19, 2017. Accessed May 8, 2018. Fan M. Plasmids 101: Multicistronic Vectors. Site Directed Mutagenesis by PCR. https://blog.addgene.org/plasmids-101-multicistronic-vectors. Accessed May 9, 2018. Postdam Bioware. Part:BBa_K929003:Design. Yeast. https://parts.igem.org/Part:BBa_K929003:Design. Published September 18, 2012. Accessed May 9, 2018. Ajo-Franklin C. Yeast. https://parts.igem.org/Yeast. Accessed May 9, 2018.