Isolation of plasmid pdf

Therefore, the non-mobile plasmids present in the sample may not be captured with this method and the bacteria not capable of growth at the specific conditions will not be included as donors. However, many resistance plasmids are conjugative, and others can be mobile when assisted by a conjugative plasmid also residing in the same bacterial cell Bennett, We are suggesting this method, not as a solution to all plasmid analysis problems, but rather as a first step in optimizing the analysis of antibiotic resistance plasmids from complex samples.

TRACA allowed for the acquisition of resistance plasmids from our sample but with similar banding patterns and resistance profiles. Therefore, it seems the expense associated with this method is not justified given the small variety of plasmids captured. The multiple displacement amplification method allowed plasmids with the greatest range of resistance profiles to be obtained from our complex cecal samples. However, it should be noted that there were also difficulties and inconsistencies with this method.

While good results were achieved using this method on Sample B shown in results , many difficulties arose while carrying out the method on both Sample A and the control sample. The DNase step can be variable and time consuming, working well after one or two treatments at some times and not working after several more at other times. This also led to further downstream complications, as the more DNase treatments the sample was subjected to, the more salt that was present in the sample.

This caused difficulties when performing electroporation, where salt concentration must be low. Plasmids now encode resistance to almost all classes of antibiotics currently in clinical use Bennett, Therefore, the study of plasmids is crucial to fight the battle against antibiotic resistance that we are currently facing.

Our comparative study shows the advantages and disadvantages of six methods for the extraction of plasmids harboring antibiotic resistance genes from complex broiler cecal samples, which can be applied to other complex environmental samples.

This will assist researchers with the selection of the best method to use in their plasmid studies. Different gram-negative bacteria other than E. The exogenous plasmid isolation method was the best for obtaining a range of multi-drug resistance plasmids in a realistic timeframe with consistent results. However, even this method only resulted in a small range of resistance plasmids being isolated. Overall, the multiple displacement amplification method provided the greatest range of resistance plasmids from the investigated cecal samples.

However, due to the inconsistencies of the results and the difficulties experienced with this method, it is not the ideal protocol to use when working with a large volume of samples under short deadlines. The commercial kits, alkaline lysis method and TRACA did not provide a wide range of resistance plasmids from our sample compared to the others tested. Therefore, the exogenous plasmid isolation method resulted in the widest range of resistance plasmids with ease of application and consistency across samples.

While this method relies on the conjugative ability of the plasmids present, it is both an efficient plasmids can be obtained in a short time-frame and effective a good range of plasmids can be acquired method which worked with all of the cecal samples tested. Therefore, we recommend the exogenous plasmid isolation method when extracting antibiotic resistance plasmids of clinical relevance from a large number of complex samples.

Ethical approval was not required for this study. The sampling was performed by Alltech from a commercial farm. These sampling techniques were in line with national regulations about animal welfare ethics. All the animals were monitored throughout the study. SD performed all experiments, data analysis, and manuscript preparation. RM executed the data analysis and prepared manuscript. FW designed the study, executed the data analysis, and prepared the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

National Center for Biotechnology Information , U. Front Microbiol. Published online Aug Author information Article notes Copyright and License information Disclaimer. This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.

Received Mar 15; Accepted Jul The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This article has been cited by other articles in PMC. Abstract The direct extraction of plasmid DNA containing antibiotic resistance genes from complex samples is imperative when studying plasmid-mediated antibiotic resistance from a One Health perspective, in order to obtain a wide representation of all the resistance plasmids present in these microbial communities. Keywords: plasmids, extraction methods, broiler, antibiotic resistance, pathogen.

Introduction The rapid rise of antibiotic resistance has led to further studies into mobile genetic elements. Materials and methods Samples The broiler cecal samples were collected from a commercial poultry production unit in the United Kingdom. Plasmid extractions and identifications Culture dependent method Cecal sample 0. Multiple displacement amplification The multiple displacement amplification method utilizes the rolling circle amplification mechanism of phi29 DNA polymerase to obtain large amounts of plasmid DNA from a complex sample.

Results Culture dependent method All cultivable bacteria grew on a non-selective rich medium and the DNA was extracted using a commercial plasmid extraction kit. Open in a separate window. Figure 1. Figure 2.

Figure 3. Table 1 Disk diffusion results of resistant transformants obtained from each of the extraction methods. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure Table 2 Transformants with identical resistance profiles from a disk diffusion assay. Tet, selected on tetracycline; Cip, selected on ciprofloxacin. B2, Higher band on gel; extracted and electroporated into E. Conclusion Overall, the multiple displacement amplification method provided the greatest range of resistance plasmids from the investigated cecal samples.

Ethics statement Ethical approval was not required for this study. Author contributions SD performed all experiments, data analysis, and manuscript preparation. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This project was sponsored by a Ph. References Arredondo-Alonso S. On the im possibility of reconstructing plasmids from whole genome short-read sequencing data.

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res.

Performance Standards for Antimicrobial Susceptibility. Plasmid diversity and adaptation analyzed by massive sequencing of Escherichia coli plasmids. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements? Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Network analyses structure genetic diversity in independent genetic worlds. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Methods 4 , 51— Current strategies for mobilome research. Strategy for purifying plasmid DNA from E. A chemical property of protein is totally different from nucleic acids; therefore, it is rather easy to separate 4.

The definitive principle for plasmid isolation: denaturation of DNA double-strand by alkaline lysis. To purify plasmid from E. Materials required.

Resuspend pellets by pipetting in and out vigorously. Overview The purification of plasmid DNA is an important part of molecular biology. In Chapter 13 of your textbook the use of plasmid DNA is discussed. Materials: 1. The Escherichia coli bacterial. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a We reasoned that the efficiency of various isolation methods might be dependent on their effects on E. Modeling resting potentials in Neurons Modeling action potentials Modeling the delayed rectifier Potassium channels Modeling the sodium ion channel and its effects on neural signaling Current Clamp protocol Voltage Clamp Protocol Understanding Frequency-Current relationship Understanding first spike latency - current relationship Voltage-Current VI plot Effects of pharmacological blockers on action potential Biochemistry Virtual Lab I Biochemistry is the study of the chemical processes in living organisms.

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