Difference between revisions of "Part:BBa K1996002:Experience"

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===Applications of BBa_K1996002===
 
===Applications of BBa_K1996002===
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EtnR1 protein from <i>Mycobacterium</i> NBB4, when expressed in <i>E. coli.</i> and purified by his-tag, binds to the EtnP promoter (<a href="https://parts.igem.org/Part:BBa_K1996000">BBa_K1996000</a>) as demonstrated by electrophoretic mobility shift assay (Figure 1). When bound by EtnR1, the EtnP DNA is less able to migrate through the agarose gel and therefore presents at a higher band size.
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EtnR1 protein from <i>Mycobacterium</i> NBB4, when expressed in <i>E. coli.</i> and purified by his-tag, binds to the EtnP promoter (BBa_K1996000) as demonstrated by electrophoretic mobility shift assay (Figure 1). When bound by EtnR1, the EtnP DNA is less able to migrate through the agarose gel and therefore presents at a higher band size.
  
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<img src="https://static.igem.org/mediawiki/2016/b/b0/T--Sydney_Australia--GelShiftFigure.png" alt="EtnR1 EMSA" width="400" height=:300"> <br>
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<img src="https://static.igem.org/mediawiki/2016/b/b0/T--Sydney_Australia--GelShiftFigure.png"> <br>
 
Figure 1. Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA.
 
Figure 1. Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA.
  

Revision as of 01:37, 21 October 2016


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Applications of BBa_K1996002

EtnR1 protein from Mycobacterium NBB4, when expressed in E. coli. and purified by his-tag, binds to the EtnP promoter (BBa_K1996000) as demonstrated by electrophoretic mobility shift assay (Figure 1). When bound by EtnR1, the EtnP DNA is less able to migrate through the agarose gel and therefore presents at a higher band size.

<img src="T--Sydney_Australia--GelShiftFigure.png">
Figure 1. Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA.

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