"

From 2011.igem.org

---

Characterization Data for BBa_K524000

Heat sensitive origin of replication (oriR101 & repA101-ts)

Abstract

oriR101 and repA101ts 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 the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).

In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, 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). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.

#Figure 1: Digestion with SpeI for confirmation of mutation success
<img src="" alt="" width="554" height="144">

Characterization

• Construct preparation for heat sensitivity test

Characterization of the oriR101&repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry’s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b E. coli strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as “ori-ts” for convenience.

• Characterization method

Qualitative test

For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 cell/7μl, ~10 cells/7μl, ~100 cells/7μl, and ~1000 cells/7μl. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at a specific incubation temperature. Four parallel sets of experiment was done, each incubated at 30°C/ 33°C/ 37°C/ 42°C. To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates

Quantitative test

To quantitatively determine the elimination rate of plasmids bearing our oriR101&repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on an of LB+ Amp plate (labeled as Amp_ori-ts) and LB- only plate (labeled as LB_ori-ts). Four copies of this experimental setup were made for overnight incubations at four different temperatures: 30°C/ 33°C/ 37°C/ 42°C. Then, single colonies formed on the plates after overnight incubation were quantified. For each set, ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was calculated. The same protocol described above was applied to the same E.coli strain (DH10B) containing only the plasmids pKD46 and pBlueScript to serve as controls.
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:
<img src="" alt="" width="554">

• Characterization result

• Qualitative test

Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.

#Figure 2: Qualitative Description of the temperature sensitivity test data.

<img src="" width="900px" align="middle">

A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region, and associated with proper functioning of 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 testing result of the unmutated oriR101&repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.

• Quantitative test

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.

<img src="" width="800px">

Conclusion

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

</td></tr></table>