Difference between revisions of "Part:BBa K902066"

 
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<partinfo>BBa_K902066 short</partinfo>
 
<partinfo>BBa_K902066 short</partinfo>
  
Encodes for the pRha, RBS site and a GFP with an LVA tag. This circuit is used to characterize expression of the rhamnose promoter with GFP containing a degradation tag. Please view our [http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#rhamnose Wiki] for more details about this component of our project.
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This component uses the rhamnose promoter (P<sub>rha</sub>) 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.  
[[Image:Calgary_RhaGFPFinal.png]]
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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.]]
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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 platforms 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. Induction of this construct was entirely dependent on levels of these regulatory proteins from the native <i>E coli.</i> genome. In future trials, we hope characterize P<sub>rha</sub> by also expressing the two control proteins on the constructs. Other teams might try placing <i>rhaS</i> and <i>rhaR</i> on the opposite side of P<sub>rha</sub> (see the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902065 P<sub>rha</sub> page] to learn of how this promoter also controls <i>rhaR</i> and <i>rhaS</i>). For the purpose of P<sub>rha</sub> and our kill system, we wish attempt constitutive expression of <i>rhaS</i> so that our system is not dependent on the presence of rhamnose to activate the kill genes. More experimentation is necessary.
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We later tested this promoter out with an <I>S7 micrococcal nuclease</i>.  This data can be found [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902084 here].
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 19:21, 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 platforms 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. Induction of this construct was entirely dependent on levels of these regulatory proteins from the native E coli. genome. In future trials, we hope characterize Prha by also expressing the two control proteins on the constructs. Other teams might try placing rhaS and rhaR on the opposite side of Prha (see the Prha page to learn of how this promoter also controls rhaR and rhaS). For the purpose of Prha and our kill system, we wish attempt constitutive expression of rhaS so that our system is not dependent on the presence of rhamnose to activate the kill genes. More experimentation is necessary.


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