Part:BBa_K4288006

pVeg-GsiB RBS-signal peptide of RpmG-ferulyol esterase

pVeg-GsiB RBS-signal peptide of RpmG-ferulyol esterase

BBa_K4288006

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

The construct used to constantly express the feruloyl esterase in order to test the enzymatic activity.

Construct design

Figure 1. map of pHY300PLK-Veg-biosensor.

Experimental approach

1.1The 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 1. 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.

1.2The feruloyl esterase-pHY300PLK fragment was obtained by PCR

Figure 3. 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 2. 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.

1.4colony 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.

Figure 5. colony PCR of Veg-biosensor recombination plasmid.

1.5Sequencing 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.

Figure 6. Sequence alignment.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]