*3.2. PCR and Sequencing*

PCR amplification of the *pldA* gene was performed using the primers PldA\_NdeI 5- - CCG**CATATG**GAAGCAACGATTGAAAAGATTC-3 and PldA\_XhoI-Rev 5- -A**CTCGAG**TT AAAGGACATCGTTCAAC-3- , which included restriction sites for NdeI and *XhoI* (in bold letters). The expected amplicon size was 833 bp. PCR amplification of the *gfp* gene was performed using the primers GFP-F 5- -CCG**CATATG**GAGAGCGACGAGAGCGGCCTGCC CG-3 and GFP-R 5- -TGAT**CTCGAG**TTATTCTTCACCGGCATCTGCATCCG-3- . The expected amplicon size was 715 bp. The PCR conditions were as follows: initial denaturation at 95 ◦C for 5 min, then 25 cycles of 94 ◦C for 15 s, 55 ◦C for 10 s, and 72 ◦C for 30 s, followed by a final extension step at 72 ◦C for 5 min. PCR-colonies was performed with standard T7-promoter/T7-terminator primers. The PCR fragments were evaluated on a 1.5% agarose gel stained with ethidium bromide. For subsequent cloning or sequencing, unincorporated primers and dNTPs were removed from PCR products with a GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sequencing was performed on a 3130xl Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions.

## *3.3. Molecular Cloning*

The plasmid vector pRSETa (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), designed for gene expression in prokaryotic cells such as *E. coli* BL21 (DE3) pLysS strain, was used. Vector and inserts (pldA, gfp, and pldA-gfp) were digested by *NdeI* and *XhoI* restrictases (Thermo Scientific). Then, inserts were ligated into vectors. The ligated products were transformed into *E. coli* TOP10- F cells (Invitrogen) and the resultant transformants were selected on carbenicillin plates and subjected to PCR-colony with T7 promoter primers. Several of the PCR-positive clones were inoculated into 3 mL test-tube cultures and allowed to grow overnight at 37 ◦C in a shaker at 200 rpm. Then, plasmid DNA were extracted from these cultures with GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Appropriate insertions of genes into the pRSETa vector were verified by DNA sequencing.
