Difference between revisions of "Part:BBa K4395013"
 
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1.Pick A, Rühmann B, Schmid J, Sieber V. Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production. Appl Microbiol Biotechnol. 2013;97(13):5815-5824. doi:10.1007/s00253-012-4474-5
 
1.Pick A, Rühmann B, Schmid J, Sieber V. Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production. Appl Microbiol Biotechnol. 2013;97(13):5815-5824. doi:10.1007/s00253-012-4474-5
 
2.Kramer L, Le X, Rodriguez M, Wilson MA, Guo J, Niu W. Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy. ACS Synth Biol. 2020;9(7):1632-1637. doi:10.1021/acssynbio.0c00290
 
2.Kramer L, Le X, Rodriguez M, Wilson MA, Guo J, Niu W. Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy. ACS Synth Biol. 2020;9(7):1632-1637. doi:10.1021/acssynbio.0c00290
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==Contribution From NJTech-China-B 2023==
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'''Group''':[https://2023.igem.wiki/njtech-china-b/ iGEM NJTech-China-B]
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'''Author''': Yao Yao
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===Characterization from iGEM23-NJTech-China-B===
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===YahK===
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YahK, an aldehyde reductase from <i>Escherichia coli</i>, catalyzes the reduction of various aldehydes to corresponding alcohols. Here, we charactered the length of the YahK gene, the molecular weight of YahK protein and the activity of YahK for future iGEM teams.
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<html lang="en">
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</head>
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<body>
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    <h2>1 Construct design</h2>
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    <h3>1.1 The transformation of palsmid pRSFDuet-YahK into <i>E. coli</i> BL21(DE3)</h3>
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    <p>
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        The gene of YahK was integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-YahK. By sequencing, the correct plasmid pRSFDuet-YahK was then transformed to <i>E. coli</i> BL21(DE3) (Figure 1). <br>
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    </p>
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1311.png" width="45%" height="45%"></p>
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    </div>
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    <div align="center">
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        <strong>Figure 1. The transformation of palsmid pRSFDuet-YahK into <i>E. coli</i> BL21(DE3)</strong>
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    </div>
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    <h3>1.2 The colony PCR of pRSFDuet-YahK in <i>E. coli</i> BL21(DE3)</h3>
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    <p>
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        The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 1500bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-YahK was successful obtained (Figure 2).<br>
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    </p>
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1312.png" width="40%" height="45%"></p>
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    </div>
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    <div align="center">
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        <strong>Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-YahK</strong>
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    </div>
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    <p>
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        M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containing plasmid pRSFDuet-YahK
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    </p>
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    <h2>2 Protein expression of YahK</h2>
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    <p>
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        The correct colony of recombinant <i>E. coli</i> BL21(DE3) containing pRSFDuet-YahK was inoculated and cultured. We transformed the plasmid pRSFDuet without YahK gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of YahK protein was correct, which was consistent with the expected molecular weight of 38.0 kDa (Figure 3).
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    </p>
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution132.png" width="40%" height="45%"></p>
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    </div>
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    <div align="center">
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        <strong>Figure3. SDS-PAGE analysis of YahK expression in <i>E. coli</i> BL21(DE3)</strong>
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    </div>
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    <p>
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        Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of <i>E. coli</i> BL21(DE3) containing pRSFDuet; lanes 3 and 4:supernatant and precipitation of <i>E. coli</i> BL21(DE3) containing pRSFDuet-YahK
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    </p>
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    <h2>3 Determination of YahK activity</h2>
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    <p>
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        YahK is an NADPH dependent aldehyde reductase. The enzyme of Yahk was purified by with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column. Subsequently, we tested its activity to catalyze 1,5-glutaraldehyde by detecting NADPH consumption with the purified YahK enzyme. The results showed that YahK can consume NADPH, indicating that YahK has active enzyme activity towards the reduction of 1,5-glutaraldehyde.
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    </p>
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    <div align="center">
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution133.png" width="45%" height="45%"></p>
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    </div>
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    <div align="center">
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        <strong>Figure 4 The enzymatic activity of purified YahK</strong>
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    </div>
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</body>
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</html>

Latest revision as of 08:57, 12 October 2023

NADPH-dependent aldehyde reductase YahK catalyzes the reduction of a wide range of aldehydes into their corresponding alcohols. Has a strong preference for NADPH over NADH as the electron donor. Cannot use a ketone as substrate. Is a major source of NADPH-dependent aldehyde reductase activity in E.coli[1]. In addition, this enzyme has been actively expressed in E.coli[2].These characteristics of RsAs attract us and thus we choose it as aldehyde reductase of our experiment.

Reference: 1.Pick A, Rühmann B, Schmid J, Sieber V. Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production. Appl Microbiol Biotechnol. 2013;97(13):5815-5824. doi:10.1007/s00253-012-4474-5 2.Kramer L, Le X, Rodriguez M, Wilson MA, Guo J, Niu W. Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy. ACS Synth Biol. 2020;9(7):1632-1637. doi:10.1021/acssynbio.0c00290

Contribution From NJTech-China-B 2023

Group:iGEM NJTech-China-B

Author: Yao Yao

Characterization from iGEM23-NJTech-China-B

YahK

YahK, an aldehyde reductase from Escherichia coli, catalyzes the reduction of various aldehydes to corresponding alcohols. Here, we charactered the length of the YahK gene, the molecular weight of YahK protein and the activity of YahK for future iGEM teams.

1 Construct design

1.1 The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)

The gene of YahK was integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-YahK. By sequencing, the correct plasmid pRSFDuet-YahK was then transformed to E. coli BL21(DE3) (Figure 1).

Figure 1. The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)

1.2 The colony PCR of pRSFDuet-YahK in E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 1500bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-YahK was successful obtained (Figure 2).

Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-YahK

M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containing plasmid pRSFDuet-YahK

2 Protein expression of YahK

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-YahK was inoculated and cultured. We transformed the plasmid pRSFDuet without YahK gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of YahK protein was correct, which was consistent with the expected molecular weight of 38.0 kDa (Figure 3).

Figure3. SDS-PAGE analysis of YahK expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet; lanes 3 and 4:supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet-YahK

3 Determination of YahK activity

YahK is an NADPH dependent aldehyde reductase. The enzyme of Yahk was purified by with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column. Subsequently, we tested its activity to catalyze 1,5-glutaraldehyde by detecting NADPH consumption with the purified YahK enzyme. The results showed that YahK can consume NADPH, indicating that YahK has active enzyme activity towards the reduction of 1,5-glutaraldehyde.

Figure 4 The enzymatic activity of purified YahK