#TITLE_ALTERNATIVE#
Instability of plasmid segregation is one of factors that influence bacterial productivity in recombinant protein synthesis. Antibiotics are usually used in bacterial fermentation process for maintaining plasmid stability and preventing contamination of other microbiological agents. However, the ris...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/20920 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Instability of plasmid segregation is one of factors that influence bacterial productivity in recombinant protein synthesis. Antibiotics are usually used in bacterial fermentation process for maintaining plasmid stability and preventing contamination of other microbiological agents. However, the risk of antibiotics release to environments and potency of genetic transfer to pathogenic bacteria causing the use of antibiotics need to be avoided. In previous studies, vector based on toxin/antitoxin selection system had been constructed that consist of two plasmids each containing CcdB toxin and CcdA antitoxin gene. This study focuses on modification and characterization of the plasmid containing CcdB gene. This study aimed to delete the pMB1 derivative ori from pUC57-Kan_ccdB, to insert cer DNA fragment into plasmid that deleted its pMB1 derivative ori, to determine arabinose optimal concentration and appropriate time for PBAD promoter induction, and to determine percentage of plasmid retention. A pair of primer was designed to recognize the outside region of pMB1 derivative ori, so all parts of the plasmid except the ori region was amplified. This deleted plasmid was named pDCS-B. Amplification product of cer DNA fragment from pDCS-A plasmid was inserted into pDCS-B to obtain pDCS-B_cer. The effect of CcdB gene expression on growth of E. coli was observed every 2 hours after arabinose induction. Percentage of plasmid retention was observed at 12 and 24 hours, without arabinose induction, at 6 and 12 hours, under arabinose induction. Deletion of pMB1 derivative ori and insertion of cer DNA fragment was performed and confirmed by nucleotide sequencing analysis. Expression of CcdB gene decreased the OD600 of E. coli after 0.2% arabinose induction at OD600 of 0.1-0.2. Insertion of cer DNA fragment proved to increase the plasmid retention by 5-10%. According to this study, pDCS-B_cer plasmid has better performance than pDCS-B. |
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