Regulatory

Part:BBa_K206001:Design

Designed by: Amelia Hardjasa   Group: iGEM09_British_Columbia   (2009-10-17)


Name: pBAD weak
Input: L-arabinose
Output: PoPS

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Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Niland et al. found that certain mutations in the AraI1 operator site increased binding of the DNA to the AraC protein [1]. We applied all of these mutations with the goal of creating a stronger version of the pBAD promoter. See pBAD Promoter Family for more details.

Source

Site-directed mutagenesis was performed on BBa_I13453 using the following primers:
Forward: 5'-PO4-TAATCTTATGGATAAAAAAGCTATGGCATAGC-3'
Reverse: 5'-PO4-GCGGATCCTACCTGACGCTTTTTATC-3'

Site-directed mutagenesis: We used the Finnzyme Phusion Site-directed Mutagenesis Kit in accordance with the manufacturer's directions.
Primers: Phosphorylated oligos were purchased from Integrated DNA Technologies (IDT) and resuspended in water.
Template: BBa_I13453 was obtained from the 2009 iGEM Spring Distribution according to Registry instructions and used as the template for site-directed mutagenesis.

References

[1] Niland P, Hühne R, and Müller-Hill B. (1996). How AraC Interacts Specifically with its Target DNAs. J. Mol. Biol. 264:667-674.