Difference between revisions of "Part:BBa K2462000"
 
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<partinfo>BBa_K2462000 short</partinfo>
 
<partinfo>BBa_K2462000 short</partinfo>
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===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.
 
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.
  
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===Verification===
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===Usage and Biology===
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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.
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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.
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The results of HPLC was depicted as following:
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https://static.igem.org/mediawiki/parts/1/14/WHU-China-Part-02.png
 
https://static.igem.org/mediawiki/parts/1/14/WHU-China-Part-02.png
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As the figure shows, green dots stand for group without RdhANP; blue dots stand for group with RdhANP. Peak areas of 2,6-DCP (appear around 6min according to the stand curve tested by stand sample 2,6-DCP) in both 0min and 120min were noted. And through the figure, the concentration change of 2,6-DCP through the process can be expressed.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Discussion===
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However, unfortunately, we are surprised to find that in the bacteria with RdhANP, even though they are induced to express RdhANP, the peak area in 120min can be even higher than it initially. At first we wondered if there are nonstandard operation during experiment, thus we did the experiment for another 2 times but nothing changed. Even the control group got the similar results. Later, we asked  the teacher in Testing Center of WHU, and he figured out the problem in some way. He told us that some proteins existed in our sample, performing the peak area together with 2,6-DCP. So it’s difficult for us to specifically quantify the sample. We were quite disappointed and tried to find another way for the analysis.
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===Reference===
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[1]Payne KA, Quezada CP, Fisher K, et al. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation. Nature. 2015;517(7535):513-516. doi:10.1038/nature13901.
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[2]Jugder B-E, Ertan H, Bohl S, Lee M, Marquis CP, Manefield M. Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation. Frontiers in Microbiology. 2016;7:249. doi:10.3389/fmicb.2016.00249.
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2462000 parameters</partinfo>
 
<partinfo>BBa_K2462000 parameters</partinfo>
 
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Latest revision as of 14:15, 1 November 2017


dehalogenase

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.

The results of HPLC was depicted as following:




WHU-China-Part-02.png

As the figure shows, green dots stand for group without RdhANP; blue dots stand for group with RdhANP. Peak areas of 2,6-DCP (appear around 6min according to the stand curve tested by stand sample 2,6-DCP) in both 0min and 120min were noted. And through the figure, the concentration change of 2,6-DCP through the process can be expressed.


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


Discussion

However, unfortunately, we are surprised to find that in the bacteria with RdhANP, even though they are induced to express RdhANP, the peak area in 120min can be even higher than it initially. At first we wondered if there are nonstandard operation during experiment, thus we did the experiment for another 2 times but nothing changed. Even the control group got the similar results. Later, we asked the teacher in Testing Center of WHU, and he figured out the problem in some way. He told us that some proteins existed in our sample, performing the peak area together with 2,6-DCP. So it’s difficult for us to specifically quantify the sample. We were quite disappointed and tried to find another way for the analysis.


Reference

[1]Payne KA, Quezada CP, Fisher K, et al. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation. Nature. 2015;517(7535):513-516. doi:10.1038/nature13901.

[2]Jugder B-E, Ertan H, Bohl S, Lee M, Marquis CP, Manefield M. Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation. Frontiers in Microbiology. 2016;7:249. doi:10.3389/fmicb.2016.00249.