Part:BBa_K5066001:Design
Xpp81Aa1
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We obtained the gene fragment from NCBI, conducted codon optimization before PCR amplification and inserting the sequence into the bacterial vector pET-28a, which includes a T7 promoter, ribosomal binding site, and a Lac operator. The pET series of expression plasmid is commonly used for recombinant protein production in E. coli, this is due to its T7 promoter and its adjacent Lac operator which helps suppress uninduced expression. The T7 promoter is a sequence of base pairs before the gene that acts as an indicator of where the RNA polymerase binds to begin the process of transcription. The ribosome binding site, represented as RBS on the vector, is a sequence of base pairs upstream of the start codon, allowing ribosome binding to the mRNA. The lac operator is an inhibitor which restricts the transcription of the gene, but the effects can be reversed by the binding of an inducer, in our project we used IPTG, which mimics an allolactose. The IPTG binds to the lac operator which induces the synthesis of protein.
Source
The source of the part is from the bacteria Bacillus thuringiensis obtained from NCBI
References
Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Communications Biology, 3(1). https://doi.org/10.1038/s42003-020-0939-8
Dubendorf, J. W., & Studier, F. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology, 219(1), 45–59. https://doi.org/10.1016/0022-2836(91)90856-2