DNA

Part:BBa_K5066000:Design

Designed by: Abner Tseng and Megan Chiao   Group: iGEM24_GEMS-Taiwan   (2024-09-13)


Cyt2Ba


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 399
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 399
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 399
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 399
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 399
  • 1000
    COMPATIBLE 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.

According to the datasheet, we first tried three annealing temperatures, 68°C, 69°C, 70°C. After running the gel electrophoresis, we found three different band sizes at each temperature. Therefore, we sent the PCR product for sequencing and used CLUSTALW to align the sequence. As shown in the figures below, the annealing temperature of 70°C had the highest aligned score of 99.87% among the three. Thus, the PCR for the Cyt2ba fragment will be conducted at 70°C.

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