Review Article

Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology

Figure 2

Comparison of three-gene deletion methods in E. coli. These methods can also be used in other enteric bacteria. The first and third methods can also be used for gene integration into the chromosome and promoter replacement for tuning gene expression. 2(a) plasmid-based method. Step 1 is construction of the deletion plasmid containing DNA fragments homologous to the target gene (h1 and h2), a selectable marker, and either a temperature sensitive or conditional replicon. Step 2 is double-crossover recombination; the plasmid cannot replicate in the host strain, and antibiotic-resistant colonies are selected. In step 3 , the FRT, replicon, and antibiotic resistance marker are removed by FLP. 2(b) Linear DNA-based method. Step 1 is construction of the linear DNA fragment by PCR (H1-P1 and H2-P2 as primers). H1 and H2 refer to short DNA fragments homologous to target gene. Step 2 is replacement of the target gene with the antibiotic resistance gene through crossover recombination with the help of Red recombinase. Step 3 is removal of FRT and antibiotic marker by FLP. 2(c) Two-stage recombination-based method developed in our lab. Steps 1 , 2, 3, and 5 describe construction of the plasmids and linear DNA fragments for the two-stage recombinations. Step 4 describes the first recombination step, in which the cat, sacB cassette is inserted into the target gene. Step 6 is the second recombination step, in which the cat, sacB cassette is removed by selection on sucrose.
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761042.fig.002b
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