Composite
1L

Part:BBa_K4654020:Design

Designed by: Ronja Friedhoff, Felix Jarecki   Group: iGEM23_TU-Braunschweig   (2023-10-08)


T7-Promoter_Spacer1-Li-+II_Riboswitch-Spacer2-5'nhaA_lacZ


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 30
    Illegal EcoRI site found at 3251
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 30
    Illegal EcoRI site found at 3251
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 30
    Illegal EcoRI site found at 3251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 30
    Illegal EcoRI site found at 3251
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 30
    Illegal EcoRI site found at 3251
    Illegal NgoMIV site found at 123
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

During the design process we made sure to include the spacer sequences up- and downstream of the riboswitch because they influence the folding of the riboswitch. We originally did not know where the RBS was located in Spacer Region 2 so we had to verify the location by asking the authors. We codon optimized the lacZ sequence for expression in E. coli.

Source

The T7 promoter is derived from the T7 phage. The riboswitch can be found in certain bacteria, the sequence we are using was created by White et. al. by creating a consenus sequence from different sequences of the riboswtich family1. lacZ is a gene of the lactose operon of E. coli and encodes for the enzyme ß-Galactosidase. The lac operon serves to regulate enzymes necessary for utilization, including ß-Galactosidase. The enzyme occurs in the natural biology of E. coli and plays a significant role in carbon metabolism. In the natural environment of E. coli, beta-galactosidase is involved in breaking down complex sugars, especially lactose, into their individual sugar units. For the design, we used the genetical sequence of the lacZ-Gene, found in E. coli K12.



References

1 White, N., Sadeeshkumar, H., Sun, A. et al. Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Sci Rep 12, 19145 (2022). https://doi.org/10.1038/s41598-022-20695-6


National Center for Biotechnology Information (NCBI).