Difference between revisions of "Part:BBa K1996002"

Latest revision as of 10:05, 21 October 2016

EtnR2 - Putative ethylene receptor in Mycobacterium NBB4

Putative ethylene receptor

Usage and Biology

EtnR2 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. EtnR2 is believed to be a regulatory protein that senses ethylene levels and phosphorylates EtnR1 protein (BBa_K1996001) which modulates expression of enzymes associated with 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 (BBa_K1996000) upon exposure to ethylene.

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.

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 56

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 Putative ethylene receptor
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+EtnR2 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. EtnR2 is believed to be a regulatory protein that senses ethylene levels and phosphorylates EtnR1 protein (BBa_K1996001) which modulates expression of enzymes associated with 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 (BBa_K1996000) upon exposure to ethylene.
+https://static.igem.org/mediawiki/2016/8/8a/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.
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