Team:BNU-China/Contribution

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   This year, due to the pandemic of 2019-nCov, we don’t have enough time to carry out experiments. As iGEMers, we want to make useful contributions for future iGEM teams, so we sorted out new information learned from literature about CLB2 promoter (BBa_K1051301) and SpCas9 (BBa_K2130013). Besides, our experiment and modeling also make useful contributions to SpCas9.

CLB2 promoter

  BBa_K1051301 is a promoter that promotes transcription at G2 phase of the yeast cell cycle. The original information on the Registry informed users about its usage principle, but it has not been documented by experiments or literature. We found the detailed transcription characters of its downstream genes from literature, which shows that the CLB2 promoter can periodically express exogenous genes sufficiently.

  What’s more, we found the specific features on the promoter sequence in another research. The region from position -362 to -131 can be used as a UAS for the promoter’s cell cycle regulation.

SpCas9

  BBa_K2130013 is a human condon optimized SpCas9. The original documents compared the editing efficiency of this part and Cas9/Cpf1. In our experiment, we expressed this protein in Cryptococcus neoformans and demonstrated that it can function well. We also built a model to predict the folding speed of Cas9, which is useful when teams want to control the function time of Cas9 precisely.

  The speed that Cas9 searches its target is also important for its function time. We found a literature showing how fast Cas9 can find the target. We also found (another literature provides a way to induce the function of Cas9 by lights very quickly.) a very fast Cas9 cutting method in literature, making CRISPR/Cas9 system more efficient for gene editing.

References

[1] Trcek, Tatjana et al. “Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast.” Cell vol. 147,7 (2011): 1484-97. doi:10.1016/j.cell.2011.11.051
[2] Maher M, Cong F, Kindelberger D, Nasmyth K, Dalton S. Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor. Mol Cell Biol. 1995;15(6):3129-3137. doi:10.1128/mcb.15.6.3129
[3] Jones, D. L., Leroy, P., Unoson, C., Fange, D., Ćurić, V., Lawson, M. J., & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science (New York, N.Y.), 357(6358), 1420–1424. https://doi.org/10.1126/science.aah7084
[4] Liu Y, Zou RS, He S, Nihongaki Y, Li X, Razavi S, Wu B, Ha T. Very fast CRISPR on demand. Science. 2020 Jun 12;368(6496):1265-1269. doi: 10.1126/science.aay8204. PMID: 32527834.