Background:Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 μg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109 , 3.21×109 , 2.32×1010 , 8.11×108 , respectively. The plasmid yields were 55 ng.μL-1, 69 ng.μL-1, 164 ng.μL-1 and 41 ng.μL-1, respectively. Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 μg.mL-1) was significantly (p < 0.01) higher than other doses.
Background:Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 μg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109 , 3.21×109 , 2.32×1010 , 8.11×108 , respectively. The plasmid yields were 55 ng.μL-1, 69 ng.μL-1, 164 ng.μL-1 and 41 ng.μL-1, respectively. Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 μg.mL-1) was significantly (p < 0.01) higher than other doses.
Entities:
Keywords:
Ampicillin; Escherichia coli; Plasmid; Protein
Authors: Martina Pasini; Alfred Fernández-Castané; Gloria Caminal; Tim W Overton; Pau Ferrer Journal: J Ind Microbiol Biotechnol Date: 2022-07-30 Impact factor: 4.258
Authors: Fernando Grijalva-Hernández; Jesús Vega-Estrada; Montserrat Escobar-Rosales; Jaime Ortega-López; Ricardo Aguilar-López; Alvaro R Lara; Ma Del Carmen Montes-Horcasitas Journal: Microorganisms Date: 2019-12-17