Device

Part:BBa_K206010

Designed by: Amelia Hardjasa   Group: iGEM09_British_Columbia   (2009-10-18)
Revision as of 03:42, 22 October 2009 by Ahardjasa (Talk | contribs)

Jammer proof of concept (J23100)

This is a proof of concept construct for the jammer system, i.e. gene knockdown via reverse promoter transcription.

Usage and Biology

What you can do with it:
If you are interested in inducible knockdown of any coding sequence, suffix it with our part BBa_K206008. The promoter of your product should be prefixed with a terminator that is capable of reverse termination, such as BBa_B0014.

Compatibility:
Chassis: Best used in the E. coli strain [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783], which has been modified to permit homogeneous pBAD promoter expression by substituting the chromosomal arabinose-dependent promoter of AraE (arabinose transporter protein) with a constitutive promoter [1]. Backbone: Has been shown to work on plasmid pSB1C3.
Reporter: Has been shown to work with reporter BBa_K145015.
Subparts: Has been shown to work with terminator BBa_B0014 and reverse promoter BBa_J44002.

Characterization

We inoculated 5mL overnight cultures of LB-Chlor with colonies from a streak plate of BBa_K206010-transformed BW27783 cells. In the morning, we diluted the cultures to an OD600 of 0.2 into 15mL LB-Chlor with or without 0.05% arabinose in 25mL flasks (duplicates). 16 hours post-arabinose induction, we measured GFP fluorescence of the cultures using a Becton-Dickinson FACSCalibur flow cytometer.

GFP fluorescence of Jammer with and without arabinose. Positive control is constitutively expressed GFP_LVA (driven by BBa_K206014). Negative control is BW27783 cells without any construct.

Induction with arabinose clearly results in knockdown of reporter expression, presumably

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 110
    Illegal NheI site found at 133
    Illegal NheI site found at 932
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 992
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 808

References

[http://www.ncbi.nlm.nih.gov/pubmed/11739756 [1]] Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.


[edit]
Categories
Parameters
None