In our previous work ( 20), pBBR1MCS2-Ts was lost from 99.99% of host E. Due to lack of knowledge about pBBR1MCS2 replication, the temperature sensitive mechanism of pBBR1MCS2-Ts is yet to be characterized. Plasmid pBBR1MCS2 is a widely used commercial plasmid and does not belong to the known broad host range IncP, IncQ, or IncW groups however, its replication mechanism remains elusive ( 19). One mutant plasmid (pBBR1MCS2-Ts) was identified that could be maintained in Escherichia coli T1 when grown at 30☌ but was lost from host cells when grown at 42☌ in the absence of antibiotic selection. To extend the utility of plasmid pBBR1MCS2, we mutated the rep gene and its promoter and terminator by error prone PCR and screened for temperature sensitivity in our pervious study ( 20). The first ORF is rep, involved in plasmid replication the second is mob, involved in mobilization and the third encodes aminoglycoside 3'-phosphotransferase, involved in kanamycin resistance ( Figure 1). It contains 3 important open reading frames (ORFs).
The commercially available plasmid pBBR1MCS2 ( 18), derived from pBBR1 and isolated from Bordetella bronchiseptica ( 19), is a mobilizable broad host range plasmid. Real time qPCR offers numerous advantages over traditional methods including saving time, rapid procedure, high sensitivity and precision, wide range of quantification, and use of small amounts of sample ( 17).īroad host range plasmids are of considerable interest due to their ability to maintain stability in various hosts and their application in recombinant DNA technology. Recently, a novel approach to determine PCN using real time quantitative PCR (qPCR) technology has been developed ( 9, 16). However, these methods are laborious, time consuming, and costly. Traditionally, PCN was analyzed by CsCl gradient centrifugation ( 11), fluorescence densitometry ( 12), high-pressure liquid chromatography ( 13), DNA hybridization ( 14), or capillary electrophoresis ( 15). The PCN is a key feature of a plasmid and is largely controlled by the origin of replication ( 10). The plasmid copy number (PCN) is defined as the number of copies of a plasmid present per chromosome in a cell ( 9). However, few of them focus on the plasmid copy number of temperature sensitive plasmid.
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A series of temperature sensitive plasmids have been developed ( 5) and applied for patents ( 6- 8). They are able to replicate at a permissive temperature in hosts but could be easily eliminated by elevating the temperature to nonpermissive conditions. Temperature sensitive modifications of plasmids are especially useful for plasmid curing ( 1), homologous recombination ( 2), protein expression ( 3), and transposon mutagenesis ( 4). Modification of plasmids to facilitate gene manipulation is an active research topic in biotechnology, because plasmid is the basic tool for carrying foreign genes into host cell. Backgroundīacterial plasmids are widely used as cloning vehicles in molecular cloning and expression. The Micro Bright Beacon™ is waterproof and corrosion, vibration, and shock resistant, so it will last in whatever weather conditions you experience.The PCN of temperature sensitive plasmid was very low at 42☌ and temperature sensitivity of the plasmid was mainly caused by the mutation of rep ORF, which subsequently affected the plasmid replication and stability.Įscherichia coli Plasmid Real Time Polymerase Chain Reaction Copy Number Method 1.
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