Difference between revisions of "Part:BBa K2462000"

Revision as of 12:51, 1 November 2017

dehalogenase

RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH.
For the verification of our enzyme, we constructed it into the vector pSPAsp-hp. Then the plasmid containing RdhANP was transformed into Bacillus magaterium through collaboration with FAFU-China. As for the expression, xylose was used as an inducer and the induction lasted for 12 hours under 37℃. After that, 800uM substrate(2,6-DCP was chosen by us as an representative according to the article) was added and interacted with our engineering bacteria for 2 hours under 17℃. Finally, our samples taken before and after the incubation were all tested through HPLC.
To be more specific, two groups of bacteria were tested simultaneously with one of them (Bacteria with RdhANP) as experimental group and the other (Bacteria without RdhANP) as a control.
The results of HPLC was depicted as following:

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 678
Illegal BglII site found at 1902
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 90
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 233

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 __NOTOC__
 <partinfo>BBa_K2462000 short</partinfo>
-===Description===
      RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH.
-===Verification===
      For the verification of our enzyme, we constructed it into the vector pSPAsp-hp. Then the plasmid containing RdhANP was transformed into Bacillus magaterium through collaboration with FAFU-China. As for the expression, xylose was used as an inducer and the induction lasted for 12 hours under 37℃. After that, 800uM substrate(2,6-DCP was chosen by us as an representative according to the article) was added and interacted with our engineering bacteria for 2 hours under 17℃. Finally, our samples taken before and after the incubation were all tested through HPLC.
      To be more specific, two groups of bacteria were tested simultaneously with one of them (Bacteria with RdhANP) as experimental group and the other (Bacteria without RdhANP) as a control.