Difference between revisions of "Part:BBa K206010"

Revision as of 15:01, 22 October 2009

Jammer proof of concept (J23100)

This is a proof of concept construct for the jammer system, i.e. gene knockdown via reverse promoter transcription. Theoretically, the presence of arabinose induces transcription from the reverse pBAD promoter. We hypothesized that this would knock down gene expression through several possible methods: 1) collision of RNA polymerase on the opposing strands; 2) hybridization of the complementary RNAs, blocking translation; 3) targeting of the hybridized RNAs for degradation by a putative degradosome.

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, almost to negative control levels.

Sequence and Features

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.