DNA

Part:BBa_K524000:Experience

Designed by: HO Yuan Heng Trevor   Group: iGEM11_HKUST-Hong_Kong   (2011-09-15)

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Applications of BBa_K524000

Characterization Data for BBa_K524000

Heat Sensitive Origin of Replication (oriR101 & repA101-ts)

Abstract

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, while temperatures higher than 42°C would cause the construct to cease its functions completely. (Datsenko, K.A. and Wanner, B.L. 2000).

In the process of its fabrication, a point mutation was introduced so as to make the construct compatible with Biobrick standard assembly requirements. Nevertheless, our characterization results do not seem to suggest great impacts to the construct's functions, causing only a slight increase in its thermosensitivity.

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 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

  • Construct Preparation for Heat Sensitivity Test
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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.

  • Characterization Methods
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    • Qualitative Test
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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; while cells contain a plasmid with the corresponding antibiotic-resistance gene and a normal replication origin, will survive and proliferate.
In our tests, serial dilution was performed on overnight cultures of ori-ts(+) cells. Dilutions were performed to give the following bacterial concentrations: 1 cell/7μl, 10 cells/7μl, 100 cells/7μl, and 1000 cells/7μl. 7μl of the abovementioned solutions where applied on LB and LB(Amp) plates. The plates were then incubated at 30°C, 33°C, 37°C, and 42°C. LB plates were prepared as a control to reflect the actual titration of E.coli in each dilution.

    • Quantitative Test
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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.
The total plasmid elimination rate with the thermosensitive replication origins (both the mutated version in pSB1AK3 and original version in pKD46) is computed by the following formula:

  • Characterization Results
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    • Qualitative Test
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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.

#Figure 2: Qualitative Description of the Temperature Sensitivity Test data.

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.


    • Quantitative test
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The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph:

#Figure 3: Quantitative Analysis of the temperature sensitivity origin.

Conclusion

This pKD46-derived oriR101 & repA101-ts displays obvious thermosensitive properties, which have seems to be intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purposes is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above.



References

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.

D. Manen, L. Caro.(1991).The replication of plasmid pSC101, Mol Microbiol. 1991 Feb;5(2):233-7.

Phillips GJ.(1995). New Cloning Vectors with Temperature-Sensitive Replication, Plasmid. 1999 Jan;41(1):78-81.


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