Constructing Recombinant Plasmids for Heterologous Expression in E. coli and Z. mobilis

Escherichia coli XL10-Gold from Invitrogen (USA), was cultured in Lysogeny broth (LB, 10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract). Medium was prepared according to the description in the Manual of Molecular Cloning [67 ]. Wild type Z. mobilis ZM4 was revived from frozen glycerol stocks in rich media with 5% glucose (RMG5: 50 g/L glucose, 10 g/L yeast extract, and 2 g/L KH₂PO₄) at 30 °C for 6–8 h without shaking. Shuttle vector pEZ15Asp includes origins of replication from both E. coli and Z. mobilis [44 (link)]. Strains and plasmids used in this study are listed in Table 3.Table 3

Strains and plasmids used in this study

Strains and plasmids	Description	Source
Strains
E. coli XL10-Gold	Ultracompetent cells (tetracycline and chloramphenicol resistant)	Invitrogen
Z. mobilis ZM4	Z. mobilis wild-type strain	Lab stock
Plasmids
pEZ15Asp	P15A_ori, Zymo_Ori, P_tet, Spe^R	[44 (link)]
pEZ15A-PlacUV5-EGFP	P15A_ori, Zymo_Ori, P_lacUV5::EGFP, Spe^R	This work
pEZ15A-PlacUV5-opmCherry	P15A_ori, Zymo_Ori, P_lacUV5::opmCherry, Spe^R	This work
pEZ-Dual	P15A_ori, Zymo_Ori, P_tet, Spe^R, P_lacUV5::opmCherry, P_tet::EGFP	This work

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Yang Y., Shen W., Huang J., Li R., Xiao Y., Wei H., Chou Y.C., Zhang M., Himmel M.E., Chen S., Yi L., Ma L, & Yang S. (2019). Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era. Biotechnology for Biofuels, 12, 52.

Publication 2019

Escherichia coli XL10-Gold

Cells Chloramphenicol E coli Frozen Glucose Glycerol Gold Lysogeny Nacl Origins of replication Plasmids Shuttle vector Strains Tetracycline Yeast

Corresponding Organization :

Other organizations : Hubei University, National Renewable Energy Laboratory

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Variable analysis

independent variables

Bacterial strain: E. coli XL10-Gold, Z. mobilis ZM4

dependent variables

Not explicitly mentioned

control variables

Culture media: Lysogeny broth (LB) for E. coli, rich media with 5% glucose (RMG5) for Z. mobilis
Incubation conditions: 30°C for 6-8 hours without shaking for Z. mobilis

controls

Positive control: Wild type Z. mobilis ZM4 strain
Negative control: Not mentioned

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