Complementation of yiaD gene in A. baumannii

The yiaD gene of AB5116 was amplified by PCR using PrimeSTAR Max DNA polymerase. The fragment was cloned into pAT03, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced expression vector in A. baumannii (57 (link)), using DNA Ligation Kit Ver.2.1 (TaKaRa), and then further transformed into DH5α to obtain the plasmid pAT03-yiaD. The competent AB5116ΔyiaD::kan cells were transformed with the target plasmid, which was confirmed by sequencing, and subsequently grown on LB agar containing carbenicillin and IPTG. Complementation of yiaD was confirmed by PCR using the yiaDqPCR primer set.

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Han L., Gao Y., Liu Y., Yao S., Zhong S., Zhang S., Wang J., Mi P., Wen Y., Ouyang Z., Zhang J., Al-Shamiri M.M., Li P, & Han S. (2022). An Outer Membrane Protein YiaD Contributes to Adaptive Resistance of Meropenem in Acinetobacter baumannii. Microbiology Spectrum, 10(2), e00173-22.

Publication 2022

DNA Ligation Kit Ver.2.1

Agar Carbenicillin Cells Dna polymerase Gene Ligation Plasmid Primer Vector

Corresponding Organization : First Affiliated Hospital of Xi'an Jiaotong University

Other organizations : Shaanxi Provincial People's Hospital, Shanxi Medical University, Second Affiliated Hospital of Xi'an Jiaotong University

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Variable analysis

independent variables

Amplification of the yiaD gene from AB5116 using PrimeSTAR Max DNA polymerase
Cloning the yiaD gene fragment into the pAT03 expression vector
Transformation of the pAT03-yiaD plasmid into AB5116ΔyiaD::kan cells

dependent variables

Growth of the transformed AB5116ΔyiaD::kan cells on LB agar containing carbenicillin and IPTG
Confirmation of yiaD complementation by PCR using the yiaDqPCR primer set

control variables

Use of the pAT03 expression vector, which is an IPTG-induced expression vector in A. baumannii
Use of DH5α cells to obtain the pAT03-yiaD plasmid
Use of LB agar containing carbenicillin and IPTG for the growth of the transformed AB5116ΔyiaD::kan cells

controls

Positive control: Confirmation of yiaD complementation by PCR using the yiaDqPCR primer set

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