Difference between revisions of "Part:BBa K3776001"

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===Characterization from iGEM23-NJTech-China-B===
 
===Characterization from iGEM23-NJTech-China-B===
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===DavB===
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L-lysine monooxygenase (DavB) is an enzyme in the 5-aminivalerate pathway. Here, we characterized the length of the davB gene, the molecular weight of DavB protein and the activity of DavB to consume lysine for future iGEM teams.
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     <div class="h3">DavB</div>
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      5-adminovalerate is produced from L-lysine through the 5-aminovalerate pathway. L-lysine monooxygenase (DavB) plays a key role in the 5-aminivalerate pathway of various microorganisms. In our experiment, DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide.
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     <h2>1 Construct design</h2>
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     <h3>1.1 The transformation of palsmid pRSFDuet-davB into <i>Escherichia coli</i> BL21(DE3)</h3>
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    <p>
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        The gene of davB was incorporated into plasmid pRSFDuet to obtain the plasmid pRSFDuet-davB. By sequencing, the correct plasmid pRSFDuet-davB was then transformed to <i>E. coli</i> BL21(DE3) (Figure 1). <br>
 
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     <div class="h3">1. Construct design</div>
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1111.png" width="45%" height="45%"></p>
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    <b>1.1 The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)</b>
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      The gene of DavB was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavB. By sequencing, the correct plasmid pRSFDuet-DavB was then transformed to E. coli BL21(DE3) (Figure 1).
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     <p class="photo-desc">Figure1The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)</p>
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        <strong>Figure 1. The transformation of palsmid pRSFDuet-davB into <i>E. coli</i> BL21(DE3)</strong>
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      <b>1.2 The colony PCR of pRSFDuet-DavB 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 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavB 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-DavB.</p>
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     <h3>1.2 The colony PCR of pRSFDuet-davB in <i>E. coli</i> BL21(DE3)</h3>  
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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-DavB</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 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-davB was successful obtained (Figure 2).<br>
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    <div class="h3">2. Protein expression of DavB</div>
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      The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB 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 DavB 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 DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1112.png" width="40%" height="45%"></p>
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     <p class="photo-desc">Figure3. SDS-PAGE analysis of DavB 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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        <strong>Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-davB.</strong>
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    <p class="photo-desc">and precipitation of E. coli BL21(DE3) containg PRSFDuet-DavB </p>
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     <div class="h3">3. Determination of DavB activity</div>
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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-davB
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      <b>3.1 Determination of DavB whole-cells catalytic activity</b>
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    <h2>2 Protein expression of DavB</h2>
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      DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB 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. Subsequently, we tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).
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     <p>
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        The correct colony of recombinant <i>E. coli</i> BL21(DE3) containing pRSFDuet-davB was inoculated and cultured. We transformed the plasmid pRSFDuet without davB 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 DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).
 
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution112.png" width="40%" height="45%"></p>
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     <p class="photo-desc">Figure4 The enzymatic activity of whole-cells</p>
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        <strong>Figure3. SDS-PAGE analysis of DavB expression in <i>E. coli</i> BL21(DE3)</strong>
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     <p class="content-paragraph">
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      <b>3.2 Detemination of crude enzyme of DavB</b>
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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-davB
 
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     <h2>3 Determination of DavB activity</h2>
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      The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB 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. Subsequently, we tested its activity by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).
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    <h3>3.1 Determination of DavB whole-cells catalytic activity</h3>
 +
    <p>
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        DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. We tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).
 
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        <p><img src="https://static.igem.wiki/teams/4800/wiki/main/contribution/contribution1131.png" width="45%" height="45%"></p>
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     <p class="photo-desc">Figure5 The enzymatic activity of crude enzyme</p>  
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        <strong>Figure 4 The enzymatic activity of the whole-cells containing DavB</strong>
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    <h3>3.2 Detemination of crude enzyme of DavB</h3>
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    <p>
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        We tested the activity of DavB by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).
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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/contribution1132.png " width="45%" height="45%"></p>
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    </div>
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        <strong>Figure 5. The enzymatic activity of crude enzyme of DavB</strong>
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Latest revision as of 10:31, 12 October 2023

DavB

DavB, L-Lysine monooxygenase, catalyzes the reaction of L-lysine to 5-aminovaleramide in the natural 5-aminovalerate pathway in Pseudomonas putida KT2440. This DavB sequence has been codon optimized for expression in Synechococcus elongatus UTEX 2973.


References

Liu, P., Zhang, H., Lv, M., Hu, M., Li, Z., Gao, C., Xu, P., & Ma, C. (2014). Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase. Scientific Reports, 4(1). https://doi.org/10.1038/srep05657

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 239
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1328
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1488
    Illegal AgeI site found at 895
    Illegal AgeI site found at 1144
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution From NJTech-China-B 2023

Group:iGEM NJTech-China-B

Author: Yao Yao

Characterization from iGEM23-NJTech-China-B

DavB

L-lysine monooxygenase (DavB) is an enzyme in the 5-aminivalerate pathway. Here, we characterized the length of the davB gene, the molecular weight of DavB protein and the activity of DavB to consume lysine for future iGEM teams.

1 Construct design

1.1 The transformation of palsmid pRSFDuet-davB into Escherichia coli BL21(DE3)

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

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

1.2 The colony PCR of pRSFDuet-davB 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 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-davB was successful obtained (Figure 2).

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

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

2 Protein expression of DavB

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davB was inoculated and cultured. We transformed the plasmid pRSFDuet without davB 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 DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).

Figure3. SDS-PAGE analysis of DavB 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-davB

3 Determination of DavB activity

3.1 Determination of DavB whole-cells catalytic activity

DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. We tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).

Figure 4 The enzymatic activity of the whole-cells containing DavB

3.2 Detemination of crude enzyme of DavB

We tested the activity of DavB by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).

Figure 5. The enzymatic activity of crude enzyme of DavB