Team:HK SSC/Improve
Improve a Previous Part or Previous Project
For part improvement, we decided to improve part BBa_K1689013
Original Part BBa_K1689013
Part BBa_K1689013 is an N-terminal fragment of β-lactamase fused with dCas9. Team iGEM15_Peking designed it to be resistance against several antibiotics. However, β-lactamase may not be applicable to each and every project. For example, in our project, the plasmid already confers Kanamycin resistance gene. β-lactamase may not be applicable in this situation. Other than using β-lactamase as a selection method, we hope to provide more options for CRISPR imaging. The GFP will allow visual confirmation of successful transformation and indicates that the dCas9 enzyme has been successfully expressed.
New Part: BBa_K3219000
dCas9 enzyme is also known as a catalytically dead Cas9 enzyme Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.
In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.
We did not add ribosome binding sites or promoters to this sequence to allow larger flexibility for users to choose the promoter and RBS that is suitable for their chassis.
References
|
[1] |
Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196. |
|
[2] |
R. M. Maier, "Bacterial Growth," in Environmental
Microbiology, Elsevier Inc, 2009, pp. 37-54. |
|
[3] |
B. G. Hall, H. Acar, A. Nandipat and M. Barlow,
"Growth Rates Made Easy," Molecular Biology and Evolution, vol.
31, no. 1, p. 232–238, 2014. |
|
[4] |
K. M. C. Tjørve and E. Tjørve, "The use of Gompertz
models in growth analyses, and new Gompertz-model approach: An addition to
the Unified-Richards family," PLoS ONE, vol. 12, no. 6, p.
e0178691, 2017. |
|
[5] |
P. R. Koya and A. T. Goshu, "Solutions of Rate-state
Equation Describing Biological Growths," American Journal of
Mathematics and Statistics, vol. 3, no. 6, pp. 305-311, 2013. |