Difference between revisions of "Part:BBa K4395013"

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===Characterization from iGEM23-NJTech-China-B===
 
===Characterization from iGEM23-NJTech-China-B===
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        <div class="h3">Yahk</div>
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     <h2>1 Construct design</h2>
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    <h3>1.1 he transformation of palsmid pRSFDuet-YahK into <i>Escherichia coli</i> BL21(DE3)</h3>
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          YahK, an aldehyde reductase from Escherichia coli, catalyzes the reduction of various aldehydes to corresponding alcohols. YahK has been found to be important in NADP/NADPH homeostasis, lipid biosynthesis, amino acid metabolism, or the formation of fuel alcohols. The broad substrate range of YahK implies its more universal function. In our experiment, YahK was used to reduce Glutaraldehyde to produce pentanediol..  
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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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         <div class="h3">Construct design</div>
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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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        <b>1.1 The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)</b>
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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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          The gene of YahK was amplified from the genome of E. coli MG1655, and 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).  
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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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        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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            <img class="earth rotatehidden" src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution11.png">
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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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        <p class="photo-desc">Figure1The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)</p>
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        <strong>Figure 2. Colony PCR results of recombinant strain that containig the plasmid pRSFDuet-YahK</strong>
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         M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg plasmid pRSFDuet-YahK
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    <h2>2 Protein expression of YahK</h2>
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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><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution132.png" width="40%" height="45%"></p>
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         <strong>Figure3. SDS-PAGE analysis of YahK expression in <i>E. coli</i> BL21(DE3)</strong>
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        Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of <i>E. coli</i> BL21(DE3) containg pRSFDuet; lanes 3 and 4:supernatant and precipitation of <i>E. coli</i> BL21(DE3) containg PRSFDuet-YahK
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    <h2>3 Determination of YahK activity</h2>
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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><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution133.png" width="45%" height="45%"></p>
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        <strong>Figure 4 The enzymatic activity of purified YahK</strong>
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          <b>1.2 The colony PCR of pRSFDuet-YahK in E. coli BL21</b>(DE3)                           
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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).                       
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        <p class="photo-desc">Figure2. Colony PCR results of recombinant strain that containig the plasmid pRSFDuet-YahK.</p>
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        <p class="photo-desc">  M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg plasmid pRSFDuet-YahK</p> 
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        <div class="h3">2. Protein expression of YahK</div>
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          The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-YahK was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. 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 class="photo-desc">Figure3. SDS-PAGE analysis of YahK expression in E. coli BL21(DE3) </p>
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        <p class="photo-desc">Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant  </p>
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        <p class="photo-desc">and precipitation of E. coli BL21(DE3) containg PRSFDuet-YahK </p>
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        <div class="h3">3. Determination of YahK activity</div>
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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 by detecting NADPH consumption with the purified YahK enzyme. The results showed that YahK can consume NADPH, indicating that YahK has active enzyme activity.
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        <p class="photo-desc">Figure4 The enzymatic activity of purified YahK </p>  
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Revision as of 03:33, 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

1 Construct design

1.1 he transformation of palsmid pRSFDuet-YahK into Escherichia 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 containig the plasmid pRSFDuet-YahK

M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg 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) containg pRSFDuet; lanes 3 and 4:supernatant and precipitation of E. coli BL21(DE3) containg 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