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Iranian Journal of Biotechnology، جلد ۱۵، شماره ۲، صفحات ۱۲۸-۱۳۴
عنوان فارسی
چکیده فارسی مقاله
کلیدواژه‌های فارسی مقاله
عنوان انگلیسی The Increase in Protein and Plasmid Yields of E. coli with Optimized Concentration of Ampicillin as Selection Marker
چکیده انگلیسی مقاله 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.
کلیدواژه‌های انگلیسی مقاله Ampicillin, Escherichia coli, Plasmid, Protein
نویسندگان مقاله صادق فیض اله زاده |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

شیرین کوهپایه |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

ایلناز rahimmansh |
department of molecular biology and genetics, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

حسین خان احمد |
department of molecular biology and genetics, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

فایزه سبزه ای |
department of molecular biology and genetics, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

مزدک گنجعلی خانی حاکمی | ganjalikhani hakemi
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

علیرضا عندلیب |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

زهرا حجازی |
department of molecular biology and genetics, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

عباس رضایی |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 81746-73461 iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

نشانی اینترنتی http://www.ijbiotech.com/article_49544_a47f7222598844d38f1a30b611d4d43e.pdf
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/441/article-441-422623.pdf
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زبان مقاله منتشر شده en
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