Difference between revisions of "Part:BBa K902066"
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[[Image:Calgary_RhaGFPFinal.png|thumb|500px|center|Top ten <i>E coli</i> expressing GFP with P<sub>rha</sub>. <i>Figure one:</i> Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars. Overnight cultures of <i>E. coli</i> with K902066 were grown in LB broth. Samples were spun down, washed and resuspended in minimal media containing different sugars, and incubated at 37<sup>o</sup>C in a 96 well plate. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP, whereas cells exposed to glucose showed minimal fluorescence similar to the sample lacking either sugar.]]
 
[[Image:Calgary_RhaGFPFinal.png|thumb|500px|center|Top ten <i>E coli</i> expressing GFP with P<sub>rha</sub>. <i>Figure one:</i> Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars. Overnight cultures of <i>E. coli</i> with K902066 were grown in LB broth. Samples were spun down, washed and resuspended in minimal media containing different sugars, and incubated at 37<sup>o</sup>C in a 96 well plate. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP, whereas cells exposed to glucose showed minimal fluorescence similar to the sample lacking either sugar.]]
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<i>Figure one</i> indicates that not only can we induce P<sub>rha</sub> with rhamnose, but that the promoter is quite tightly regulated with minimal leaky expression without the rhamnose inducer. This makes it an excellent candidate for a killswitch construct, as it would allow for minimal expression of kill genes when when activators are not present. P<sub>rha</sub> is more tightly regulated as opposed to other expression platform already present in the registry. Consider, for example, the [https://parts.igem.org/Part:BBa_R0010 LacI] promoter which show significant leaky expression without IPTG inducer (see Figure 4C where we showed this leaky expression on our [http://2012.igem.org/Team:Calgary/Project/FRED/Reporting electrochemical reporting page]).  
 
<i>Figure one</i> indicates that not only can we induce P<sub>rha</sub> with rhamnose, but that the promoter is quite tightly regulated with minimal leaky expression without the rhamnose inducer. This makes it an excellent candidate for a killswitch construct, as it would allow for minimal expression of kill genes when when activators are not present. P<sub>rha</sub> is more tightly regulated as opposed to other expression platform already present in the registry. Consider, for example, the [https://parts.igem.org/Part:BBa_R0010 LacI] promoter which show significant leaky expression without IPTG inducer (see Figure 4C where we showed this leaky expression on our [http://2012.igem.org/Team:Calgary/Project/FRED/Reporting electrochemical reporting page]).  
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Note that this construct does not involve the use of either [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902069 <i>rhaR</i>] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902068 <i>rhaS</i>] regulatory elements for the rhamnose system. In the
  
  

Revision as of 17:51, 28 October 2012

pRha-B0034-K082003

This component uses the rhamnose promoter (Prha) to control GFP with an LVA tag. We used this circuit to characterize how the rhamnose promoter is affected by rhamnose, glucose, and lack of these sugars. Please view the [http://2012.igem.org/Team:Calgary/Notebook/Protocols/Prha_Characterization protocol] which we used to conduct this experiment. On our [http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#rhamnose Wiki], we discuss how we implemented this expression platform for control of our kill switch.

Top ten E coli expressing GFP with Prha. Figure one: Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars. Overnight cultures of E. coli with K902066 were grown in LB broth. Samples were spun down, washed and resuspended in minimal media containing different sugars, and incubated at 37oC in a 96 well plate. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP, whereas cells exposed to glucose showed minimal fluorescence similar to the sample lacking either sugar.


Figure one indicates that not only can we induce Prha with rhamnose, but that the promoter is quite tightly regulated with minimal leaky expression without the rhamnose inducer. This makes it an excellent candidate for a killswitch construct, as it would allow for minimal expression of kill genes when when activators are not present. Prha is more tightly regulated as opposed to other expression platform already present in the registry. Consider, for example, the LacI promoter which show significant leaky expression without IPTG inducer (see Figure 4C where we showed this leaky expression on our [http://2012.igem.org/Team:Calgary/Project/FRED/Reporting electrochemical reporting page]).


Note that this construct does not involve the use of either rhaR or rhaS regulatory elements for the rhamnose system. In the


We later tested this promoter out with an S7 micrococcal nuclease. This data can be found here

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 910