Part:BBa_K3776002
DavA
DavA, 5-aminovaleramide amidohydrolase, catalyzes the reaction of 5-aminovaleramide to 5-aminovalerate in the natural 5-aminovalerate pathway in Pseudomonas putida KT2440. This DavA 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution From NJTech-China-B 2023
Group:iGEM NJTech-China-B
Author: Yao Yao
Characterization from iGEM23-NJTech-China-B
5-Aminovalerateis produced from L-lysine through the 5-aminovalerate pathway. 5-aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. In our experiment, DavA catalyzes 5-aminovaleramide into 5-aminovalerate.
1.1 The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)
The gene of DavA was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavA. By sequencing, the correct plasmid pRSFDuet-DavA was then transformed to E. coli BL21(DE3) (Figure 1). 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).
Figure1The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)
1.2 The colony PCR of pRSFDuet-DavA 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 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavA was successful obtained (Figure 2).
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA 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 DavA 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 DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 3).
Figure3. SDS-PAGE analysis of DavA 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-DavA
3.1 Determination of DavA whole-cells catalytic activity
DavA catalyzes 5-aminovaleramide into 5- aminovalerate.The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA 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 production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 4).
Figure4 The enzymatic activity of whole-cells
3.2 Detemination of crude enzyme of DavA
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA 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 production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 5).
Figure5 The enzymatic activity of crude enzyme
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