Part:BBa_K3407016
B. subtilis decarboxylase (bsdBCD) with inducible T7 promoter, RBS and T7 terminator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 531
Illegal AgeI site found at 121
Illegal AgeI site found at 373
Illegal AgeI site found at 1202
Illegal AgeI site found at 1292
Illegal AgeI site found at 1494 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composte part encodes for the reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis (bsdBCD) under the T7 promoter and terminator. bsdBCD is an enzyme that has been reported to decarboxylate phenolic acids, including vanillate and 4-hydroxybenzoate. It has also been described to perform the carboxylation reaction for substrates such as guaiacol and phenol, therefore performing reversible reactions for these compounds. This enzyme is composed of three subunits B, C and D with respective sizes 22.5 kDa, 53 kDa and 8.5 kDa [1]. (Figure 1). The presence of all three gene products is strictly necessary for its activity [2].
Experimental results
In order to determine the expression of our designed construct, we incubated E. coli BL21 (DE3) transformed with the plasmid pTWIST_bsdBCD, at 37ºC until the OD600 reached ~ 0.6. We induced the cultures with a final IPTG concentration of 0.1 mM and incubated them at 37ºC, taking samples at different time points. We performed the same experiment with different conditions of induction (0.2 and 0.5 mM IPTG and incubation at 20ºC). The total protein content of the cells from both experiments was analysed by SDS-PAGE electrophoresis (Figure 2).
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Figure 2:SDS-PAGE of total protein content E. coli BL21 (DE3) transformed with pTWIST_bsdBCD. Cells were induced with 0.1 mM IPTG and samples were taken at different time points. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 1h 3h 4h (1, 3 or 4 hour after induction), ON (overnight). All the samples used corresponded to the same OD600. . -
Figure 3:SDS-PAGE of total protein content E. coli BL21 (DE3) transformed with pTWIST_bsdBCD.Cells were induced with 0.5 and 1 mM IPTG and samples were taken at different time points. The non-induced sample is a negative control to check leaky expression. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600. . - Lupa, B., Lyon, D., Shaw, L. N., Sieprawska-Lupa, M., & Wiegel, J. (2008). Properties of the reversible nonoxidative vanillate/4-hydroxybenzoate decarboxylase from Bacillus subtilis. Canadian journal of microbiology, 54(1), 75–81.
- Lupa, B., Lyon, D., Gibbs, M. D., Reeves, R. A., & Wiegel, J. (2005). Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases. Genomics, 86(3), 342–351.
As can be seen in Figure 2 and 3, bands corresponding to the molecular weights of ≈ 50 kDa and ≈ 20 kDa can be observed, corresponding to subunits C and B respectively. Subunit D is 8.5 kDa and is not shown on the gel, as it possibly migrated off the gel due to its small size.
References
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