Difference between revisions of "Part:BBa K206010"

 
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__NOTOC__
 
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<partinfo>BBa_K206010 short</partinfo>
 
<partinfo>BBa_K206010 short</partinfo>
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This is a proof of concept construct for the jammer system, i.e. gene knockdown via reverse promoter transcription.
 
This is a proof of concept construct for the jammer system, i.e. gene knockdown via reverse promoter transcription.
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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''What you can do with it:''
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<br>If you are interested in inducible knockdown of any coding sequence, suffix it with our part <partinfo>K206008</partinfo>. The promoter of your product should be prefixed with a terminator that is capable of reverse termination, such as <partinfo>B0014</partinfo>.
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''Compatibility:''
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<br>
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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 [[Part:BBa_K206010#References|[1]]].
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Backbone: Has been shown to work on plasmid <partinfo>pSB1C3</partinfo>.
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<br>
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Reporter: Has been shown to work with reporter <partinfo>K145015</partinfo>.
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<br>
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Subparts: Has been shown to work with terminator <partinfo>B0014</partinfo> and reverse promoter <partinfo>J44002</partinfo>.
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===Characterization===
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We inoculated 5mL overnight cultures of LB-Chlor with colonies from a streak plate of <partinfo>K206010</partinfo>-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.
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[[Image:J23100-Jammer_Complete.png|center|frame|GFP fluorescence of Jammer with and without arabinose. Positive control is constitutively expressed GFP_LVA (driven by <partinfo>K206014</partinfo>). Negative control is BW27783 cells without any construct.]]
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Induction with arabinose clearly results in knockdown of reporter expression, presumably
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K206010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K206010 SequenceAndFeatures</partinfo>
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===References===
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[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.
  
  

Revision as of 03:42, 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.

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.