Part:BBa_K3776001

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

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