The Increase in Protein and Plasmid Yields of E. coli with Optimized Concentration of Ampicillin as Selection Marker

Abstract

is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in , but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid. Alterations of protein expression and plasmid yields of in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized. The expression cassette of codon optimized EGFP for was synthesized in pUC57. The pUC57-GFP was transformed into. 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 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve. 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 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×10 , 3.21×10 , 2.32×10 , 8.11×10 , respectively. The plasmid yields were 55 ng.μL, 69 ng.μL, 164 ng.μL and 41 ng.μL, respectively. Protein and plasmid yields of 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) was significantly (p < 0.01) higher than other doses.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

“…4 A, from the pre-induction sample to the end of the fermentation, the reference strain presented a significant percentage of unhealthy and dead cells even under uninduced conditions. The high proportion of damaged or dead cells present before the induction stage is probably due to the presence of the antibiotic in the medium, which is used to maintain plasmid stability (Feizollahzadeh et al, 2017 ). The percentage of damaged cells remained relatively constant through growth (3–5%).…”

High Kanamycin Concentration as Another Stress Factor Additional to Temperature to Increase pDNA Production in E. coli DH5α Batch and Fed-Batch Cultures

“…The pDNA production consisted in an initial growth phase to accumulate a high biomass concentration at low temperatures (30-37 • C), followed by a temperature upshift (42-45 • C) to induce a high pDNA replication rate (revised by Jaén et al [11]). For this reason, the temperature level used were 30 and 42 • C. Likewise, selection marker concentration can drastically affect the plasmid yield and antibiotic concentration levels, therefore, they were selected according to ones reported by Feizollahzadeh et al [34]. The statistical analysis was performed at 99% confidence level using the software Minitab 17.…”

Host-aware RNA-based control of synthetic microbial consortia

“…However, it is important to note that the expression of these control circuits themselves can impose a burden on cell growth, leading to reduced achievable ranges of population composition 47 . This undesirable effect, along with the use of toxins, antimicrobials, and burdensome control strategies, calls for the exploration of new approaches that can be upscaled to industrial production without compromising growth rates, and in turn bioproduction capacity [49][50][51][52][53] . This work proposes an RNA-based control system to regulate the population composition in a two-member E. coli coculture.…”