Part:BBa_K4288006
pVeg-GsiB RBS-signal peptide of RpmG-ferulyol esterase
pVeg-GsiB RBS-signal peptide of RpmG-ferulyol esterase
Feruloyl esterase (FAE, EC 3.1.1.73), also known as cinnamate esterase or cinnamic acid hydrolase, is an extracellular carboxylesterase that mainly degrades cell wall
Profile
Name: pVeg-GsiB RBS-signal peptide of RpmG-feruloyl esterase
Base Pairs: 1085 bp
Origin: E.coli
Properties: constitutive expression of feruloyl esterase
Usage and biology
polysaccharides or arabinose in pectin or an ester bond between galactose or hydroxycinnamic acid. Our project aims to couple the concentration of heavy metals in the sample to be tested with the color in the reaction solution through the catalysis of FAE to produce a color product, so as to visually display the concentration of metal ions in the sample. Therefore, we designed a plasmid pHY300PLK-Pveg-Biosensor for constitutive expression FAE in Bacillus subtilis to test the enzymatic activity of FAE to provide enzyme data for our biosensor.
Construct design
The pHY300PLK-Veg-biosensor construct used to constantly express the feruloyl esterase in order to test the enzymatic activity.
1.1 The promotor veg fragment was amplified by PCR.
The promoter veg DNA fragment was amplified by primers Veg-F and Veg-R. The length of promoter Veg is 96 bp. In addition, we used the super-fidelity Pfu DNA polymerase and its extension time of Pfu is 2 min/kb. The extension time of PCR depends on the length of target DNA and DNA polymerase. Thus, the thermal cycler program set as 20 seconds for extension time of PCR at 72℃. The PCR products were analyzed by 1.5% agar gel electrophoresis. The electrophoresis result showed correct band in figure 2. The band of promoter Veg were extract by gel extraction kit according to the protocol. The DNA fragment concentration was determined using NanoDrop. The result showed the Veg fragment concentration is 3 ng/μl at a final volume of 40 μl.
1.2 The feruloyl esterase-pHY300PLK fragment was obtained by PCR
The biosensor-pHY300PLK fragment was amplified by PCR using the primers pVector-F and pVector-R. The template is biosensor-pHY300PLK plasmid which was extracted from E.coli Top10. Oving to the length of biosensor-pHY300PLK fragment (5902 bp), we prolong the extension time of PCR for 340 seconds at 72℃ although we used the same Pfu DNA polymerase. The electrophoresis result showed correct band in figure 3. The band biosensor-pHY300PLK of were extract by gel extraction kit according to the protocol. Biosensor-pHY300PLK concentration is 3 ng/μl at a final volume of 40 μl, which was measured by NanoDrop.
1.3 LB agar plate containing the single colonies of Veg-biosensor
The Veg fragment and pHY300PLK vector ligated using Gibson assembly method. Then, the recombinant plasmid was transformed into the competent cells DH10. The bacteria spread to the selection plate with antibiotic and incubated at 37 ℃ overnight.
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]
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