Difference between revisions of "Part:BBa K3407000"
 
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<partinfo>BBa_K3407000 SequenceAndFeatures</partinfo>
 
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===Experimental results===
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==Experimental results==
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In order to determine the expression of our designed construct, we incubated <i>E. coli</i> 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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In order to determine the expression of our designed construct, we incubated <i>E. coli</i> 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 (figure 2). 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 3).
  
 
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   <li style="display: inline-block;"> [[File:T--TUDelft--bsdBCD_1.png|thumb|none|800px|<b>Figure 2:</b>SDS-PAGE of total protein content <i>E. coli</i> BL21 (DE3) transformed with pTWIST_bsdBCD. Cells were induced with 0.1 mM IPTG and samples were taken at different time points.
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   <li style="display: inline-block;"> [[File:T--TUDelft--bsdBCD_1.png|thumb|none|800px|<b>Figure 2:</b>SDS-PAGE of total protein content <i>E. coli</i> 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.  
  
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. ]] </li><li style="display: inline-block;"> [[File:T--TUDelft--bsdBCD_2.png|thumb|none|800px|<b>Figure 2:</b>SDS-PAGE of total protein content <i>E. coli</i> BL21 (DE3) transformed with pTWIST_bsdBCD.Cells were induced with 0.5 and 1 mM IPTG and samples were taken at different time points.
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. ]] </li><li style="display: inline-block;"> [[File:T--TUDelft--bsdBCD_2.png|thumb|none|800px|<b>Figure 3:</b>SDS-PAGE of total protein content <i>E. coli</i> 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.  
  
 
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As can be seen in Figure 2A and B, 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.  
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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.
  
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===References===
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==References==
 
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Latest revision as of 00:36, 28 October 2020

Reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from B. subtilis (bsdBCD)


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 477
    Illegal AgeI site found at 67
    Illegal AgeI site found at 319
    Illegal AgeI site found at 1148
    Illegal AgeI site found at 1238
    Illegal AgeI site found at 1440
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and biology

This basic part encodes for the reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis (bsdBCD). 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 (figure 2). 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 3).

  • 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. .

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

Ordered List

  1. 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.
  2. 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.