E coli jm109 cells

DNA fragments with a 600 bp size, amplified from the environmental DNA by PCR, were excised from the agarose gel, digested with the BlpI and BglII restriction endonucleases, and ligated into the pTV-Taq' plasmid predigested with the BlpI and BglII restriction endonucleases. The ligation mixtures were introduced into E. coli JM109 cells (TaKaRa Bio.), and 20 clones were picked independently from the transformants for each amplification. Plasmid DNAs were extracted from these clones, and the nucleotide sequences of the DNA inserts were determined by CEQ2000XL DNA analysis system (Beckman Coulter, USA).

The partial 18S rRNA gene and the ITS region were amplified from 14 samples that tested positive via RLB using the primer pairs P1/P2 and ITSF/ITS2, respectively (Table 2). PCR amplification of the 18S rRNA gene and ITS sequences was performed in a total volume of 50 μl, with 10 μl of 5 × TransStart FastPfu Buffer, 5 μl of a 2.5 mM dNTP Mixture, 0.1 μM of each primer, 1 μl of TransStart FastPfu DNA Polymerase (Takara Biotechnology, Dalian, China), 2.5 μl of genomic DNA, and double distilled water. The conditions for PCR amplification of the 18S rRNA gene were as follows: an initial denaturation step at 95°C for 2 min; 35 cycles of denaturation for 20 s at 95°C, annealing for 20 s at 55°C, and extension for 45 s at 72°C; and a final extension step of 5 min at 72°C. The PCR amplification conditions for the ITS region were almost the same as for the 18S rRNA gene, except that the annealing temperature used was 54°C in this step. The PCR products were purified using the Easypure Quick Gel Extraction Kit (TransGen Biotech, Beijing, China). The purified amplicons were cloned into the pMD19-T vector (Takara Biotechnology, China), which was then transformed into E. coli JM109 cells (TaKaRa Biotechnology, China) according to the manufacturer's instructions. Three positive colonies of each sample were selected for sequencing (ABI PRISM 377 DNA sequencer).

We introduced random mutations into YGFPuv and YGFPdp sequences, which are both YGFP derivatives obtained from the 1^st screening, using a previously described method with certain modifications [20 (link)]. The YGFP variant genes were PCR amplified using ExTaq (Takara Bio Inc) according to the manufacturer’s instructions, with primers 5ʹ-TTGAATTCATGACAACCTTCAAAATCGAG and 5ʹ-AATTAA-GCTTCTACATGTCTCTTGGGGCGC. The purified PCR product (1.9 μg) was digested into small fragments using DNase I for 20 min at 25°C. DNA fragments between 50 and 300 bp were separated on a 2% agarose gel, and subsequently mixed for PCR at 10–30 ng/μl DNA concentration. The mixed DNA fragments were then diluted 50-fold in a new PCR mixture and re-amplified with the primers described above. The PCR products were cloned into the pEGFP vector employing the Hind III and EcoR I sites as described above and transformed into E. coli JM109 cells (Takara Bio Inc). For each cycle of DNA shuffling, approximately 30,000 colonies were obtained. The brightest 20–40 colonies were selected and pooled for the next cycle of visual screening under a UV light box, as described above. We conducted 3 cycles of this DNA shuffling procedure and sequenced the selected YGFP mutants’ genes, as described above.