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Published on: October 14, 2011
The enhancement of M13 phage titration by optimizing the origin of replication
Mohammad Hossein Darvishali1, Mahmood Fadaie1, Hossein Khanahmad1
1Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Background And Purpose:
M13KO7, a modified M13 phage variant, carries the p15A replication origin and Tn903 kanamycin resistance gene. This study aimed to optimize M13KO7's replication by substituting the p15A origin with the higher-copy pMB1 origin (500-700 copy numbers).
Experimental Approach:
A 6431-nucleotide fragment from the M13KO7 plasmid lacking the p15A replication origin and kanamycin resistance gene was amplified using a long polymerase chain reaction (PCR). The modified M13AMB1 plasmid was created by adding adenine to the 3' ends of this fragment and ligating it to the pMB1-containing fragment using T/A cloning. Afterward, to prepare the phage, pM13AMB1 was transformed into E. coli TG1 bacteria, and then, using the PEG-NaCl precipitation, the modified phage was propagated. The modified phage titer was determined utilizing the serial dilution and the qPCR methods, compared with the M13KO7 phage.
Findings/Results:
The results showed that in the serial dilution method, the titers of modified phage and M13KO7 phage were 4.8 × 1014 and 7 × 1012 pfu/mL, respectively. Besides, the phage titer calculated by the qPCR method for the modified phage was equal to 1.3 × 109 pfu/mL, whereas it was 4.08 × 108 pfu/mL for the M13KO7 phage.
Conclusion And Implications:
This study provides evidence that replication origin replacement led to a significant increase in phage titers. It highlights the importance of replication optimization for molecular biology applications.
Insights
Replacing the p15A origin with the pMB1 origin significantly increased M13KO7 phage titers. This optimization enhances phage replication for molecular biology applications.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- M13KO7 is a modified M13 phage variant.
- It utilizes the p15A replication origin and a kanamycin resistance gene.
- The study aimed to enhance M13KO7 replication.
Purpose of the Study:
- To optimize M13KO7 phage replication.
- To substitute the native p15A origin with the higher-copy pMB1 origin.
- To increase phage yield for molecular biology applications.
Main Methods:
- A DNA fragment from M13KO7 was amplified using long PCR.
- The p15A origin was replaced with the pMB1 origin via T/A cloning, creating M13AMB1.
- Phage was propagated in *E. coli* TG1, and titers were determined by serial dilution and qPCR.
Main Results:
- The modified phage (M13AMB1) showed significantly higher titers than M13KO7.
- Serial dilution yielded 4.8 × 10^14 pfu/mL for M13AMB1 vs. 7 × 10^12 pfu/mL for M13KO7.
- qPCR indicated 1.3 × 10^9 pfu/mL for M13AMB1 vs. 4.08 × 10^8 pfu/mL for M13KO7.
Conclusions:
- Replication origin replacement successfully increased phage titers.
- The pMB1 origin enhances M13KO7 phage replication.
- Optimizing replication origins is crucial for improving phage-based molecular tools.
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