1. BBa_K524000 –oriR101 & repA101-ts: This heat sensitive origin of replication has been shown to be functional at 30°C. Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.
Characterization Data for BBa_K524000
Heat Sensitive Origin of Replication (oriR101 & repA101-ts)
oriR101 & repA101-ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat-labile protein product of repA101-ts, which loses activity significantly at 37°C. Hence when bacteria harboring plasmids with this origin are incubated at 37°C, the activity of this origin would decrease. Temperatures higher than 42°C would cause the construct to cease its functions completely. (Phillips GJ, 1995).
Construction of This Part
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cut site originally present inside the construct, specifically between the promoter and CDS of the ori101R & repA101-ts. The mutation has been successfully confirmed by restriction digestion (Figure 1) and nucleotide sequencing.
#Figure 1: Digestion with SpeI for Confirmation of Successful Mutation
Characterization of the oriR101 & repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry.org's standard assembly pSB1AK3 plasmid. The enzymes PstI and SmaI were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. After the blunt end treatment, the linearized plasmid was self-ligated and transformed into E. coli DH10b. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used for plasmid verification. The final construct used for thermosensitivity test is referred to as “ori-ts” for convenience.
To test the thermosensitivity of the origin, we employed the plasmid-loss assay. When the cells were incubated in non-permissive temperatures (i.e. temperatures above 37°C), the plasmid containing the antibiotic-resistance gene will be lost and no growth of cells harboring ori-ts will be seen on the antibiotic-containing plates (here we used ampicillin with working concentrations of 150μg/ml); while cells that contain a plasmid with the corresponding antibiotic-resistance gene and a normal replication origin will survive and proliferate.
To quantitatively determine the elimination rate of plasmids bearing our oriR101 & repA101-ts, (approximated) equal numbers of ori-ts-harboring bacterial cells were spread on LB plates (labeled as LB_ori-ts) and LB (Amp) plates (labeled as Amp_ori-ts). Four replicas of this experimental setup were prepared for overnight incubations at four different temperatures: 30°C, 33°C, 37°C, and 42°C. Then, colonies formed on the plates after overnight incubation were quantified. For each set, the ratios of colonies on Amp_ori-ts plates vs. LB_ori-ts plates were calculated. The same protocol described above was applied to the same E. coli strain (DH10b) containing only the plasmids pKD46 and pBlueScriptKS+ to serve as controls.
Several sets of serial dilution tests were done at 30°C, 33°C, 37°C and 42°C. The evidence and results are shown below.
For each temperature point in the gradient, the most concentrated O/N culture was applied on the leftmost-side, and serially diluted by 10-fold towards the rightward direction.
A robust growth of ori-ts(+) cells was observed when the cells were incubated at 30°C. This robustness (indicated by the density of bacterial colonies in each droplet, and associated with the proper functioning of the plasmid's heat sensitive origin) steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation, and near-complete loss of function was observed at 37°C. Comparing this trend with the tested result of the original oriR101 & repA101-ts in pKD46 (partial loss of function observed at 37°C, and near-complete loss of function at 42°C), the mutated one shows higher thermosensitivity.
The result of the quantitative thermosensitive test and trend of the % elimination of ori-ts is shown by the following graph:
#Figure 3: Quantitative Analysis of the temperature sensitivity origin.
Datsenko KA, Wanner BL.(2000).One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.
Experiments and Results