用TthI酶切pNRW33，去除一个pigB内部片段,用klenow酶填补5’端overhang，然后将质粒骨架自连接得到pNRW34。 pigBΔ等位基因克隆到pKNG101的SmaI位点，构建了标记交换结构体pNRW41。为了构建 pigDΔ等位基因，将pNRW35用NruI/NdeI酶切后补平末端并自连接得到pNRW36，将pNRW36用BamHI酶切后克隆到pKNG101中得到pigDΔ标记交换构建pNRW40。用引物对pigM的5’和3’侧翼区域进行扩增，用SalI-BamHI和BamHI-SpeI酶切，三向连接到标记交换载体pKNG101中，生成pNRW52。用引物扩增pigK的5’和3’侧翼区，用SalI-HindIII和HindIII-SpeI酶切，与pKNG101三向连接，得到pNRW51。用引物NW49-Cdelr和Cdelf-NW50扩增pigC的5¢和3¢侧翼区域，用SalI-BamHI和BamHI-SpeI酶切，与pKNG101三向连接，得到pNRW54。用引物扩增pigA的5’和3’侧翼区，用SalI-BamHI和BamHI-SpeI酶切，与pKNG101三向连接，得到pNRW58。pigGD标记交换质粒pNRW53也以类似的方式创建。用引物扩增5’和3’侧翼区，后续PCR产物用SalI-HindIII和HindIII-ApaI酶切。用NW39- NW37和NW38-NW21PCR扩增pigED的5’和3’侧翼区，分别用BamHI-HindIII和HindIII-SalI酶切。

上述两者都天然拥有SalI位点，用T4连接酶将PCR产物连接。

参考文献：Williamson NR, Simonsen HT, Ahmed RA, Goldet G, Slater H, Woodley L, Leeper FJ, Salmond GP. Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces. Mol Microbiol. 2005 May;56(4):971-89.