Team:Lethbridge/Improve

Improved Parts

**Highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)

Original Part: BBa_E1010
Submitted by: Antiquity
Designed by: Drew Endy
Improvement rationale: A 6X Histidine tag and linker was placed on the N-terminal end for purification purposes. This was done to ensure that we could use the red fluorescent protein as a control for activity assays, expression, and measurement standardization of our insulin constructs. The His tag allows for in vitro control testing while the linker ensures no steric hindrance that might prevent appropriate folding of the protein. This construct was also improved in a different way through codon optimization for expression in C. reinhardtii. In this case, the construct has the addition of the psaA 5’UTR (BBa_K2148000) and rbcL 3’UTR (BBa_K2148016).

Proinsulin (with modified protease recognition sites)

Original Part: BBa_K1328003
Originally Submitted by: iGEM14_Tsinghua
Originally Designed by: Lingfeng Hou
Improvement rationale: This part was improved by adding the RFP fusion and including additional sequences to ensure proper protein function. The ATG site on the proinsulin was removed to prevent protein synthesis of just the insulin coding sequence. As well, another furin cut site and linker was added in between the RFP and proinsulin coding sequences. Finally, a cell-penetrating peptide (a protein transduction domain) was added to help uptake into the intestinal epithelial cells for entry into the bloodstream. This allows for RFP to not interfere with the proinsulin protein function as well as allowing for cleavage of RFP from the proinsulin peptides once exposed to the furin enzyme in target cells. We also provided a promoter (BBa_J23119), RBS (BBa_J61100), and terminator (BBa_B0014) for the E.coli construct (which was submitted into the registry as BBa_K3237016) and a psaA 5’UTR (BBa_K2148000) and rbcL 3’UTR (BBa_K2148016) for the C. reinhardtii construct (which was submitted into the registry as BBa_K3237017).