Constructing ptxD Gene Expression Vectors

All
primers used in this study are listed in Table 2. Three different ptxD genes
from P. stutzeri,^{22 (link)}P. aeruginosa,^{27 (link)} and K. pneumoniae(28 (link)) were tried. The ptxD genes were expressed based on the pXMJ19 plasmid under the Peftu promoter. The Peftu promoter were
amplified from the pJYS3_crtYf plasmid using primers
*Peftu-F/*Peftu-R. The ptxD_Pst, ptxD_Pae, and ptxD_Kpn gene fragments (with
sequences homologous to plasmid pXMJ19) were codon-optimized and synthesized
by Nanjing Tsingke Biological Technology Co., Ltd. The Peftu promoter and each of the ptxD genes were ligated
into BamHI-linearized pXMJ19 plasmid, generating recombinant plasmid
pXMJ19*ptxD_Pst, pXMJ19*ptxD_Pae, and pXMJ19*ptxD_Kpn, respectively. The ligation
was accomplished through homologous recombination using the ClonExpress
Ultra One Step Cloning Kit (Vazyme) according to the manufacturer’s
protocol.

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Lei M., Peng X., Sun W., Zhang D., Wang Z., Yang Z., Zhang C., Yu B., Niu H., Ying H., Ouyang P., Liu D, & Chen Y. (2021). Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum. ACS Omega, 6(15), 10160-10167.

Publication 2021

ClonExpressUltra One Step Cloning Kit

Biological Codon Genes Homologous recombination P aeruginosa Plasmid Pneumoniae Primers

Corresponding Organization : Zhengzhou University

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Variable analysis

independent variables

Three different ptxD genes from P. stutzeri, P. aeruginosa, and K. pneumoniae

dependent variables

Expression of the ptxD genes based on the pXMJ19 plasmid under the Peftu promoter

control variables

The Peftu promoter amplified from the pJYS3_crtYf plasmid using primers Peftu-F and Peftu-R
The ptxD gene fragments (ptxDPst, ptxDPae, and ptxDKpn) were codon-optimized and synthesized

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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