Regulatory

Part:BBa_K3866000

Designed by: Venetios Michelioudakis Anna Patri   Group: iGEM21_Thessaly   (2021-09-14)
Revision as of 11:45, 23 September 2021 by Venetios (Talk | contribs) (→‎Usage and Biology)


araC-ParaBAD:RBS GB compatible with A1-B2



Usage and Biology

The PBAD promoter, derived from the arabinose operon andis an inducible promoter for arabinose. The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death.(Kallunki, Barisic, Jäättelä and Liu, 2019). The araC gene, is a regulatory gene and is located upstream of the L-arabinose operon, encodes a positive regulatory protein AraC, required for L-arabinose utilization in Escherichia coli. The araC gene has a constitutive promoter.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The CDS has position A1-B2

Figure 1.The overhangs of this part in the GoldenBraid Grammar.
Figure 2. The level 0 module : pupd2- araC:ParaBAD (illustration from SnapGene)

Verification of Cloning

Fig.3:(U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ PstI ,Expected bands: 2029+1996, Positive result : YES

Source

From the iGEM Distribution Kit 2019.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1148
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965


References

  • Kallunki, Barisic, Jäättelä and Liu, 2019. How to Choose the Right Inducible Gene ExpressionSystem for Mammalian Studies?Cells, 8(8), p.796.

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