Difference between revisions of "Part:BBa K902066"

Revision as of 17:07, 28 October 2012

pRha-B0034-K082003

This component uses the rhamnose promoter (P_rha) 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 P_rha. Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars in M9 minimal media. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP whereas cells exposed to glucose minimal similar to the sample lacking either sugar.

This results were very promising. This graph indicates that not only can we induce induce expression rhamnose, but the promoter is quite tightly regulated with little leaky expression over a basal level. This makes it an excellent candidate for a killswitch construct, as it would allow for very little leaky expression of any kill genes when not wanted and an easy way of inducing the kill gene expression. 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

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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|thumb|500px|center|Top ten <i>E coli</i> expressing GFP with P<sub>rha</sub>. Fluorescence output (We can see a large increase with 0.2% rhamnose, 0.5% rhamnose whereas there is no GFP expression in the cells incubated with glucose as compaed to our control.]]
+[[Image:Calgary_RhaGFPFinal.png|thumb|500px|center|Top ten <i>E coli</i> expressing GFP with P<sub>rha</sub>. Fluorescence output was measured in response to different concentrations of glucose, rhamnose, and neither of these sugars in M9 minimal media. The 0.2% rhamnose and 0.5% rhamnose showed induction of GFP whereas cells exposed to glucose minimal similar to the sample lacking either sugar.]]
 This results were very promising.  This graph indicates that not only can we induce induce expression rhamnose, but the promoter is quite tightly regulated with little leaky expression over a basal level.  This makes it an excellent candidate for a killswitch construct, as it would allow for very little leaky expression of any kill genes when not wanted and an easy way of inducing the kill gene expression.  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]