Construction of Prokaryotic Expression Vector of Cucumber LOX2 Gene

The method of inserting the cloned foreign gene into a suitable vector and then introducing it into E. coli to express a large number of proteins is generally called prokaryotic expression. Prokaryotic expression usually uses E. coli as the expression strain. Because the expression of recombinant proteins in E. coli is beneficial to growth and control, and has the characteristics of simple operation and low cost, it is a common method for expressing foreign genes in molecular biology. The first step in prokaryotic expression of foreign genes is to construct the foreign genes to be expressed into prokaryotic expression vectors. The LOX/HPL combined catalytic pathway is the only metabolic pathway for the production of cucumber aromatic substances.

Principle

Expression vectors play a very important role in genetic engineering. Prokaryotic expression vectors are usually plasmids. A typical expression vector should have a promoter, multiple cloning site, termination codon, fusion tag, replicon, selection marker reporter gene, etc. When constructing prokaryotic expression vectors, you must learn to read the plasmid map. First look at the position of Ori to understand the type of plasmid (prokaryotic / eukaryotic / shuttle plasmid). Secondly, look at the selection marker and decide which selection marker to use. Finally, look at the multiple cloning site (MCS), which has a single cutting site for multiple restriction enzymes. If a foreign gene is inserted at these sites, it will lead to the inactivation of a certain selection marker gene (such as LacZ), which facilitates screening.

Here we take the construction of the prokaryotic expression vector pET-30a(+) as an example to introduce the construction process of the prokaryotic expression vector. Use analysis software such as Premier 5 to analyze the cucumber LOX2 gene that has been cloned into the cloning vector pMD18-T. After comparing the open reading frames, the restriction endonucleases SalI and NotI were used to double-digest pMD18-T-LOX2 and the prokaryotic expression vector pET-30a(+) at the same time, and the LOX2 gene with enzyme cutting sites was recovered. Mix them at appropriate concentrations and ligate them with T4 DNA ligase to obtain the prokaryotic expression plasmid pET-30a(+)-LOX2 containing the LOX2 target gene. After the host bacteria were transformed and amplified, the recombinant plasmid was digested and identified by PCR.

Procedures

1. Digestion and recovery of pMD18-T-LOX2 and pET-30a(+)

Take two 250 µL microcentrifuge tubes, add reagents according to the system in Table 1 and Table 2, mix well, centrifuge briefly, and digest overnight at 37°C.

Table 1. Enzyme Digestion System of pMD18-T-LOX2

Table 2. Enzyme Digestion System of pET-30a(+)

After the reaction is completed, use 0.8% agarose gel for electrophoresis and serve as a negative control when loading the sample. After verifying that the enzyme digestion is correct, cut the gel and recover it, and the purified product will be used immediately or temporarily stored at -20°C. The obtained fragments were LOX2 gene fragment and pET-30a(+) vector fragment respectively.

2. Ligation reaction

Add the following reagents in sequence to a 250 µL microcentrifuge tube according to Table 3, mix and centrifuge briefly. The ligation reaction was carried out for 1h or overnight.

Table 3. Ligation System of pET-30a(+)-LOX2

3. Heat shock transformation

Add 20 µL of the ligation product to a 1.5 mL tube in an ice bath, then add 50 mL of DH5a competent cell suspension, pipe gently with the pipette tip to mix, and let stand on ice for 30 min. The transformation product was heat-shocked in a 42°C water bath for 90 s and immediately cooled on ice for 3 min. Then add 800 µL LB liquid culture medium to the tube, and incubate at 37°C, 180 r/min with shaking for 50 min until the bacterial solution is slightly turbid.

Take 20-100 µL of the above bacterial solution and spread it on the plate. After the bacterial solution is completely absorbed by the culture medium, incubate it overnight at 37°C.

4. Screening

Uniform white colonies can be seen on Kan-resistant plates cultured overnight. Use a pipette tip to pick 5 to 10 colonies, add them to 1 mL of Kan-resistant LB liquid culture medium, and incubate at 37°C with shaking at 180 r/min for 3-5 h until the bacterial liquid becomes turbid. Take 1 µL of each bacterial solution for PCR verification. If the verification is correct, sequencing can be performed for further verification.

Note:

• When selecting restriction sites, first analyze the restriction sites on the cloned fragments. The selection of restriction sites should avoid the restriction sites on the cloned fragments.
• Prokaryotic expression vectors usually have fusion tags. When constructing, special attention should be paid to the base pairing problem in gene fusion to avoid causing frameshift mutations.
• When constructing, if the target gene has the enzyme cutting sites required for construction, if other enzymes cannot be selected, you can consider introducing isocaudomers into the target gene, such as BamH I and BgZ ll, EcoR I and Mun I, etc.
• Sometimes the prokaryotic expression vector is constructed correctly, but it may not be able to express the target protein. This may be related to the type of protein. You can try to change to a different prokaryotic expression vector.

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