Difference between revisions of "Part:BBa K1996001"

 
 
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Putative transcription factor for enzymes involved in ethylene degradation pathway in Mycobacterium NBB4
 
Putative transcription factor for enzymes involved in ethylene degradation pathway in Mycobacterium NBB4
  
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===Usage and Biology===
 
===Usage and Biology===
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EtnR1 is a native protein of <i>Mycobacterium</i> NBB4. This sequence has been codon-optimised for expression in <i>E. coli.</i> where expression has been achieved in a T7 system with pGro7 chaperone protein (Figure 1) with protein identity confirmed by mass spectrometry. EtnR1 is believed to be a regulatory protein that is phosphorylated by EtnR2 (BBa_K1996002) when EtnR2 binds to ethylene. EtnR1 then binds to the (BBa_K1996000), a putative promoter regulating expression of enzymes involved in ethylene metabolism.  We suspect that together, EtnR1 and EtnR2 represent a two-component regulatory system responsible for activating gene expression from the putative promoter <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1996000">EtnP</a>  upon exposure to ethylene.
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https://static.igem.org/mediawiki/2016/8/8a/T--Sydney_Australia--EtnR1R2expression.png
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<b>Figure 1. SDS-PAGE sizing of EtnR1 and EtnR2 expressed following 6 hour IPTG induction. Cellular lysates were isolated from BL21 (DE3) E. coli. with pGro7 chaperone plasmid expressing pET-EtnR1 or pET-EtnR2, and separated electrophoretically on 12% SDS-PAGE. The bands around 66-70 kDa and 25-28 kDa respectively were trypsin-digested and analysed using mass spectrometry. (a) Isolated EtnR1 lysates. (b) Isolated EtnR2 lysates.</b>
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EtnR1 binding to EtnP DNA has been demonstrated in an electrophoretic mobility shift assay (EMSA), where it was found that EtnR1 binding to EtnP notably inhibits the migration of the DNA through the gel, indicating a strong binding affinity between EtnR1 protein and EtnP DNA (Figure 2).
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<img src="https://static.igem.org/mediawiki/2016/b/b0/T--Sydney_Australia--GelShiftFigure.png"><br>
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<b>Figure 2.  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. </b>
  
 
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Latest revision as of 19:50, 30 October 2016


EtnR1 - DNA-binding protein for EtnP in Mycobacterium NBB4

Putative transcription factor for enzymes involved in ethylene degradation pathway in Mycobacterium NBB4

Usage and Biology

EtnR1 is a native protein of Mycobacterium NBB4. This sequence has been codon-optimised for expression in E. coli. where expression has been achieved in a T7 system with pGro7 chaperone protein (Figure 1) with protein identity confirmed by mass spectrometry. EtnR1 is believed to be a regulatory protein that is phosphorylated by EtnR2 (BBa_K1996002) when EtnR2 binds to ethylene. EtnR1 then binds to the (BBa_K1996000), a putative promoter regulating expression of enzymes involved in ethylene metabolism. We suspect that together, EtnR1 and EtnR2 represent a two-component regulatory system responsible for activating gene expression from the putative promoter EtnP upon exposure to ethylene. T--Sydney_Australia--EtnR1R2expression.png Figure 1. SDS-PAGE sizing of EtnR1 and EtnR2 expressed following 6 hour IPTG induction. Cellular lysates were isolated from BL21 (DE3) E. coli. with pGro7 chaperone plasmid expressing pET-EtnR1 or pET-EtnR2, and separated electrophoretically on 12% SDS-PAGE. The bands around 66-70 kDa and 25-28 kDa respectively were trypsin-digested and analysed using mass spectrometry. (a) Isolated EtnR1 lysates. (b) Isolated EtnR2 lysates.

EtnR1 binding to EtnP DNA has been demonstrated in an electrophoretic mobility shift assay (EMSA), where it was found that EtnR1 binding to EtnP notably inhibits the migration of the DNA through the gel, indicating a strong binding affinity between EtnR1 protein and EtnP DNA (Figure 2).

<img src="T--Sydney_Australia--GelShiftFigure.png">
Figure 2. 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.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 150
    Illegal AgeI site found at 1359
  • 1000
    COMPATIBLE WITH RFC[1000]