Regulatory

Part:BBa_K4059009:Design

Designed by: Ann-Sofie Nielsen   Group: iGEM21_SDU-Denmark   (2021-10-20)
Revision as of 00:51, 22 October 2021 by AnnSofie1 (Talk | contribs) (→‎Design Notes)


T7Lac Promotor and RBS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This is a inducible promoter system, which is a mutant T7 Lac promoter. The T7 promoter system allows induction of gene expression with isopropyl-β-D-1-thiogalactopyranoside (IPTG) in E. coli strains with the bacteriophage T7-polymerase in their genome (such as ER2566 or in general BL21(DE3) derivates). The mutant T7 Lac promoter is to be used together with the lacI, encoding the LacI repressor protein. Consideration: There is choosen a medium inducible promoter system based on the former litterature where a high inducible promoter will not work in the cells.

Characterisation: In our project, the mutant T7lac promoter is combined with 5 of our genes (PsiD, PsiK, PsiM, PsiH and CPR) and placed into a plasmid containing LacI. The expression of the genes controlled by the mutant T7lac promoter is tested using Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR). The results shows that the gene expression is induced by IPTG. It increases over time and the highest expression is seen 2 hours post-induction. The induction also confirms the functionality of the LacI used (BBa_I732820), since our ER2566 strain does not naturally overexpress LacI. Therefore, the addition of a functional LacI by cloning is necessary for the T7lac promoter to be sufficiently repressed in the absence of IPTG.

Source

Source: https://pubmed.ncbi.nlm.nih.gov/31550507/ and https://www.nature.com/articles/srep11301

References