Composite

Part:BBa_K3407016

Designed by: Alicia Rodriguez Molina   Group: iGEM20_TUDelft   (2020-10-23)

B. subtilis decarboxylase (bsdBCD) with inducible T7 promoter, RBS and T7 terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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].

  • Figure 1:Genes of the three subunits of vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis.

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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