Engineering Success
Overview
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 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.
Figure 1. map of pHY300PLK-Veg-biosensor
1.1 The promotor veg fragment was amplified by PCR.
Figure 2. gel electrophoresis result of Veg fragment M: DNA ladder; lane1-2 promoter veg fragment
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
Figure3. gel electrophoresis result of feruloyl esterase-pHY300PLK fragment lane1: DNA ladder;
lane2: feruloyl esterase-pHY300PLK fragment
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.
Figure 4. LB agar plate for recombinant plasmid
1.4 colony PCR
We picked up 16 colonies for performing colony PCR. Colony PCR system comprised of primers pVeg-verf-up and pVeg-verf-dn, Taq Master mix (DNA polymerase, buffer, loading, etc.), and colonies. The electrophoresis result showed all selected colonies have correct band, figure 4. Thus, we sent No. 1, 3, 5, and 7 to DNA sequencing. The M representative maker, 1-16 representative colonies containing the recombinant plasmid.
Figure5. colony PCR of Veg-biosensor recombination plasmid
1.5 Sequencing of Veg-biosensor recombination plasmid
The sequence alignment showed that there is no mutation or mismatch. Thus, we chose the No.3 for subsequent assay.
Figure6. Sequence alignment
Functional Test
In order to confirm the protein expression and secret into the culture medium, the plasmid pHY300PLK-Pveg-Biosensor which can consistently express feruloyl esterase(FAE) was transformed into Bacillus subtilis and contand tested in different cultured time. Because FAE catalyzes the decomposition of the substrate methyl ferulate, and the enzyme activity of FAE can be obtained by detecting the decline rate of methyl ferulate at 340 nm. The chemical equation is as follow:
feruloyl-polysaccharide + H2O = ferulate + polysaccharide
The results showed that the enzymatic activity of FAE increased with the prolongation of culture time, indicating that the expression of enzyme increased with the increase of culture time.
Figure 7. The activity of ferulyol esterase in different culture time.
Learn
We have already collected the enzyme activity data from our experiments. FAE has the effect of catalyzing methyl ferulate to ferulate and polysaccharide. At the same time, since we detected the enzymatic activity of the protein in the supernatant of the culture medium, this shows that our protein can indeed be expressed in Bacillus subtilis and secreted to the outside of the cell, which verifies that our design idea is correct. It has laid a solid foundation for the cadmium-inducible promoter(Pcad) and the detection of cadmium ion concentration.
