Figure 1.
Schema of the two-step gene synthesis method.
In the first step, the fragments overlapped with each other was separately assembled by assembly PCR with the outside oligonucleotides as primers; and then, in the second step, were assembled into the full-length gene by overlap extension PCR with 5'- and 3'-end outside primers.
Figure 2.
Sequence comparison between the original and the codon optimized genes.
Dots in figure represent the same nucleotides between original and optimized genes. (A) Original and optimized ROL gene, and (B) Original and optimized phyA gene.
Figure 3.
Secondary structure of the first 100-150 bp of original and optimized mRNA generated by the software RNAfolder.
A. Structure of original ROL mRNA with the MFE is −27.3 kcal/mol and (B) structure of codon optimized ROL mRNA with the MFE is −24.5 kcal/mol; (C) Structure of original PhyA mRNA with the MFE is −67.8 kcal/mol and (D) structure of codon optimized PhyA mRNA with the MFE is −45.9 kcal/mol.
Figure 4.
Schema of the two-step ROL gene synthesis method.
Full length ROL gene was firstly divided into two overlapped fragments, F1 (473 bp) and F2 (380), and were assembled by assembly PCR separately; Then F1 and F2 was assembled into full length ROL gene by overlap extension PCR (A). (B) PCR products of F1, F2 and full-length ROL gene.
Figure 5.
Schema of the two-step phyA gene synthesis method.
Full length phyA gene was divided into four fragments (F1, F2, F3 and F4) with the end overlapped with each other and assembled by assembly PCR separately. In the second step, these four fragments were assembled into a full length gene by overlap extension PCR (A). (B) PCR products of F1, F2, F3, F4 and full-length phyA gene.
Figure 6.
Expression profiles of yeast recombinants carrying original and codon optimized ROL gene (ROL-syn).
(A) SDS-PAGE analysis of protein in fermentation broth after methanol induced for 48 h. About 20 μL protein solution per sample was loaded into the gel stained by coomassie blue G-250 after running. Protein content (B) and lipase activity (C) of fermentation broth of yeast recombinants carrying original and codon optimized ROL genes.
Figure 7.
Expression profiles of yeast recombinants carrying original and codon optimized PhyA gene (phyA-syn).
(A) SDS-PAGE analysis of protein in fermentation broth after methanol induced for 48 h. About 20 μL protein solution per sample was loaded into the gel which gone to silver stain after running; Protein content (B) and phytase activity (C) of fermentation broth from yeast recombinants carrying original and codon optimized phyA genes.
Table 1.
Comparison of different methods used for gene synthesis.