Team UFlorida Contribution
Due to limited wet lab time, team UFlorida has taken the opportunity to contribute to iGEM by adding current literature of bio-parts. We found iGEM Pittsburgh 2018 Base editor recording system such an interesting project that we decided to research more about the topic and contribute to their circuit by uploading bioparts found from papers we think will help future teams. We believe these parts can help improve the overall gene expression of the system, making the system more robust at producing an output.
For this project Team UFlorida believes this recording base excision mechanism can be improved by using a different promoter from the PlacO promoter. The Ptac promoter(BBa_K3910997) is a hybrid promoter combining the -35 trp operon region and -10 region of lacUV5. The promoter is IPTG inducible. Based on Voigt's 2021 lab paper on plasmid and promoter quantifiable activity, pTac shows to be a strong inducible promoter, reaching its highest transcript copy number at around 100 uMolar concentration of promoter. This system was coupled with a strong B0064 Ribosome Binding Site:BBa_B00 64. A strong ribosome binding site(BBa_K3910998) will allow the machinery of E.coli to bind to a sequence that due to its strong nature, will have better binding affinity to the ribosome and as such better protein expression as shown in the paper.
Figure 1. X axis shows different promoters used, Y axis shows gene expression of reporters.
Ptac promoter induced by IPTG levels
We also believe that by adding a riboJ ribozyme (BBa_K3424025) sequence downstream of the Ptet-O right before the CDS of the system sites will also allow for better expression of the Base Editor system as well. Insulators like riboJ work by creating hairpin formations in the untranslated region of a circuit, allowing the desired CDS sequence to be better expressed by reducing gene expression issues that can arise, especially using heterologous genetic circuits.
Shao, B., Rammohan, J., Anderson, D. A., Alperovich, N., Ross, D., & Voigt, C. A. (2021, March 5). Single-cell measurement of plasmid copy number and promoter activity. Nature News. Retrieved October 19, 2021, from https://www.nature.com/articles/s41467-021-21734-y#Sec21.
A plasmid toolbox for controlled gene expression across ... (n.d.). Retrieved October 19, 2021, from https://www.researchgate.net/publication/352397681_A_plasmid_toolbox_for_controlled_gene_expression_across_the_Proteobacteria