Part:BBa_K4347012:Design
Bst fusion with Sso7d and point mutations for enhanced thermal stability codon optimized for E.coli
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 971
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Point mutations were made for thermal stability to account for fluctuations in the portable heating device, as temperature fluctuations typically oscillate about the desired set point temperature when using electronic circuits. The basis of our research was based off of Taq polymerase, which is a structural homologue to Bst. In a study by Raghunathan & Marx[1], it was found that only 25% of the point mutations made in the fingers domain of Taq resulted in a PCR active polymerase whereas over 70% and 60% of the mutations in the thumb and palm domains resulted in a PCR active polymerase. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst.
The purpose of the DNA binding protien is to enhance processivity of the polymerase to result in more amplification product to yield a greater endpoint signal in our indicator via magnesium ion depletion to LAMP byproduct pyrophosphate.
Source
- Sso7d: https://parts.igem.org/Part:BBa_K3790001
- (GGGGS)4 linker: https://parts.igem.org/Part:BBa_K3117004
- Bst: https://parts.igem.org/Part:BBa_K4347010
References
1. Raghunathan, G., & Marx, A. (2019, January 24). Identification of thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Nature News. Retrieved July 12, 2022, from https://www.nature.com/articles/s41598-018-37233-y#Fig6