Name: testing the role of multicopy plasmids in the evolution of antibiotic resistance
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This work was supported by the Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016): grants CP15-00012, PI16-00860, and CIBER (CB06/02/0053), co-financed by the European Development Regional Fund &#39;&#39;A way to achieve Europe&#39;&#39; (ERDF). JAE is supported by the Atracci&#243;n de talento program of the government of the region of Madrid (2016-T1/BIO-1105) and the I+D Excelencia of the Spanish Ministerio de Econom&#237;a, Industria y Competitividad (BIO2017-85056-P). ASM is supported by a Miguel Servet Fellowship from the Instituto de Salud Carlos III (MS15/00012) co-financed by The European Social Fund &#34;Investing in your future&#34; (ESF) and ERDF.

1. Construction of the Experimental System Encoding Antibiotic Resistance Gene
Note: Here E. coli MG1655 was used as the recipient strain of the plasmid- or chromosome-encoded antibiotic resistance gene. The antibiotic resistance gene is encoded in the chromosome or a multicopy plasmid in an otherwise isogenic strain (Figure 1).
<ol>
	<li>Insertion of the antibiotic resistance gene in the &#955; phage integration site (attB)<sup class="...

<ol>
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We present a new protocol combining molecular biology, experimental evolution and deep DNA sequencing designed to investigate the role of multicopy plasmids in the evolution of antibiotic resistance in bacteria. Although this protocol combines techniques from different fields, all the methods required to develop it are simple, and can be performed in a regular microbiology laboratory. The most critical steps in the protocol probably are the construction of the model system strains and the reconstruction of the mutations ...

Antibiotic resistance in bacteria is a major health problem1. At a fundamental level, the spread of antibiotic resistance in pathogenic bacteria is a simple example of evolution by natural selection2,3. Put simply, the use of antibiotics generates selection for resistant strains. A key problem in evolutionary biology, therefore, is to understand the factors that influence the ability of bacterial populations to evolve resistance to antibiotics. Selection experiments have emerged as a very powerful tool to investigate the evolutionary biology of bacteria, and this field has produced incredible insights into a wide range of evolutionary problems4,5,6. In experimental evolution, bacterial populations initiated from a single parental strain are serially passaged under defined and tightly controlled conditions. Some of the mutations that occur during the growth of these cultures increase bacterial fitness, and these spread through the cultures by natural selection. During the experiment, samples of the populations are periodically cryogenically preserved to create a non-evolving frozen fossil record. A wide number of approaches can be used to characterize evolving bacterial populations, but the two most common methods are fitness assays, that measure the ability of evolved bacteria to compete against their distant ancestors, and whole genome sequencing, that is used to identify the genetic changes that drive adaptation. Following pioneering work by Richard Lenski and colleagues7,8, the standard approach in experimental evolution has been to challenge a relatively small number of replicate populations (typically &#60;10) with adapting to a new environmental challenge, such as new carbon sources, temperature, or a predatory phage.
Infections caused by antibiotic resistant bacteria become a big problem when resistance is high enough that it is not possible to increase antibiotic concentrations to lethal levels in patient tissues. Clinicians are therefore interested in what allows bacteria to evolve resistance to high doses of antibiotic that are above this threshold antibiotic concentration, the clinical breakpoint. How to study this experimentally? If a small number of bacterial populations are challenged with a high dose of antibiotic, as in a Lenski-style experiment, then the most likely outcome is that the antibiotic will drive all of the populations to extinction. At the same time, if the dose of antibiotic that is used is low, below the minimal inhibitory concentration (MIC) of the parental strain, then it is unlikely that the bacterial populations will evolve clinically relevant levels of resistance, especially if resistance carries a large cost. One compromise between these two scenarios is to use an &#34;evolutionary rescue&#34; experiment9,10,11. In this approach, a very large number of cultures (typically &#62;40) is challenged with doses of antibiotics that increase over time, typically by doubling antibiotic concentration every day12. The hallmark of this experiment is that any population that does not evolve increased resistance will be driven to extinction. Most populations that are challenged in this way will be driven extinct, but a small minority will persist by evolving high levels of resistance. In this paper, we show how this experimental design can be used to investigate multicopy plasmid contribution to the evolution of resistance.
Bacteria acquire resistance to antibiotics through two principal routes, chromosomal mutations, and acquisition of mobile genetic elements, mostly plasmids13. Plasmids play a key role in the evolution of antibiotic resistance because they are able to transfer resistance genes between bacteria by conjugation14,15. Plasmids can be divided into two groups according to their size and biology: &#34;small&#34;, with high copy number per bacterial cell and &#34;large&#34;, with low copy number16,17. The role of large plasmids in the evolution of antibiotic resistance has been extensively documented because they include conjugative plasmids, which are key drivers of the dissemination of resistance and multi resistance among bacteria15. Small multicopy plasmids are also extremely common in bacteria17,18, and they often code for antibiotic resistance genes19. However, the role of small multicopy plasmids in the evolution of antibiotic resistance has been studied to a lesser extent.
In a recent work, we proposed that multicopy plasmids could accelerate the evolution of the genes they carry by increasing gene mutation rates due to the higher gene copy number per cell12. Using an experimental model with E. coli strain MG1655 and the &#946;-lactamase gene blaTEM-1 it was shown that multicopy plasmids accelerated the rate of appearance of TEM-1 mutations conferring resistance to the third-generation cephalosporin ceftazidime. These results indicated that multicopy plasmids might play an important role in the evolution of antibiotic resistance.
Here, we present a detailed description of the method we have developed to investigate the multicopy plasmid-mediated evolution of antibiotic resistance. This method has three different steps: first, insertion of the gene under study either in a multicopy plasmid or the chromosome of the host bacteria. Second, use of experimental evolution (evolutionary rescue) to assess the potential of the different strains to adapt to the selective pressure. And third, determining the molecular basis underlying plasmid-mediated evolution using DNA sequencing and reconstructing the suspected mutations individually in the parental genotype.
Finally, although the protocol described here was designed to investigate the evolution of antibiotic resistance, one can argue that this method could be generally useful to analyze the evolution of innovations acquired by mutations in any multicopy plasmid-encoded gene.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermocycler</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">C1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporator</td>
			<td style="width:167px;">BiorRad</td>
			<td style="width:119px;">1652660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NanoDrop 2000/2000c</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td>Determine DNA quality measuring the ratios of absorbance 260nm/280nm and 260nm/230nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:167px;">Memmert</td>
			<td style="width:119px;">UF1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator (shaker)</td>
			<td style="width:167px;">Cole-Parmer Ltd</td>
			<td style="width:119px;">SI500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis power supply</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1645070</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis chamber</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1704405</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pippettes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">725020, 725050, 725060, 725070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Multi-channel pippetes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">728220, 728230, 
			728240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plate reader Synergy HTX</td>
			<td style="width:167px;">BioTek</td>
			<td style="width:119px;">BTS1LF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inoculating loops</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8388</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:119px;">351172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:119px;">DF0446-17-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB agar</td>
			<td style="width:167px;">Fisher scientific</td>
			<td style="width:119px;">BP1425-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion Polymerase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F533S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Gibson Assembly</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:119px;">E2611S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Resctriction enzymes</td>
			<td style="width:167px;">Fermentas FastDigest</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Antibiotics</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Plasmid extraction kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">A1120</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNeasy Blood &#38; Tissue Kits</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryotubes</td>
			<td style="width:167px;">ClearLine</td>
			<td style="width:119px;">390701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates (-80&#186;C storage)</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">249945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuantiFluor dsDNA System</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">E2670</td>
			<td>Quantification of DNA concentartion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1613100</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50x TAE buffer</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1610743</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 Polynucleotide Kinase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EK0031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA Ligase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EL0014</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the role of multicopy plasmids in the evolution of antibiotic resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

In our previous work, the evolution the &#946;-lactamase gene blaTEM-1 towards conferring resistance to the third generation cephalosporin ceftazidime12 was investigated. This gene was selected because, although TEM-1 does not confer resistance to ceftazidime, mutations in blaTEM-1 can expand the range of activity of TEM-1 to hydrolyze cephalosporins such as ceftazidime29. Mutations in antibiotic resis...

Watch this Scientific Journal Video about Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance at JoVE.com

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

The overall goal of this experimental method is to test the role of multicopy plasmids in the evolution of antibiotic resistance. This method can help to answer key questions in the field of microbiology such as the role of multicopy plasmids in the evolution of bacterial innovations. The main advantage of this technique is that it only requires basic molecular biology methods and a simple experimental design.It's really cool. First, construct the e coli MG1655 strain that will carry the antibody resistance gene in the chromosome in the lambda phage in equation side ATTB. To do so, PCR amplify the 500 base paralong regions on both sides of the ATTB site.Then PCR amplify the antibiotic resistance gene. Then use isothermal assembly at 50 degrees Celsius for 30 minutes to fuse the homology regions to the PCR product. After isothermal assembly, transfer one microliter of the product to 40 microliters of electrocompetent MG1655 cells into a two millimeter cuvette.Then electroporate the cells at 4 degrees Celsius and 2.5 kilovolts. After electroporation, transfer the cells in one milliliter of LB broth with 0.2%arabinose. Then pipette the LB broth containing cells to a microfuge tube.Shake the tube intensely for two hours at 30 degrees Celsius. After two hours, plate 100 microliters of the culture on the LB agar plate, supplemented with antibiotics. Then spin down the remaining cells.And re-suspend the pellet in 100 microliters of fresh LB broth. Re-plate these cells on another LB agar plate. Then incubate the plates at 42 degrees Celsius overnight.The following day, PCR amplify the clones to verify the construct using external primers ybhC extern and ybhB extern. Then confirm the amplicon size through the agarose shell electrophoresis. Parallelly, conduct DNA sequencing to verify the sequence of the inserted gene.To construct the strain to carry the antibody resistance gene in the multicopy plasmid, first PCR amplify the antibiotic resistance gene blaTEM-1. After the PCR is completed, phosphorylate the amplified product using T4 polynucleotide kinase. Next, use high-fidelity polymerase and oligonucleotides PBAD FINV and PBAD RINV, to PCR amplify the PBAD plasmid backbone.Then ligate the two PCR fragments using T4 ligase. Next, electroporate 40 microliters DH5 alpha cells with 1 microliter of the ligation product. Then select the appropriate antibiotic for the antibiotic resistance gene and the plasmid backbone on agar plates.Then perform plasmid extraction using a DNA extraction kit, following the manufacturer's instructions. Then perform sanger sequencing of the resistance gene of interest to confirm the absence of any possible mutations before the evolution experiments. After verification of the sequence, electroporate the plasmid in the e coli strain MG1655 to finally obtain the plasmid carrying the MG1655 strain.The most critical steps in this protocol are the construction of the model systems strains and the reconstruction of the mutations observed during experimental evolution. First, streak the MG1655 strains of interest on the LB agar plates. Then add 200 microliters of LB medium in each well of a 96 well plate.Next, inoculate 48 isolated colonies of each strain in individual wells of the plate. After inoculation, incubate the plate overnight at 37 degrees Celsius on a shaker at 200 revolutions per minute. Again, inoculate two microliters of the culture from each well with the founder population to a new 96 well plate with 198 microliters of LB medium in individual wells.This will start the evolutionary rescue experiment. Incubate the plate at 37 degree Celsius on a shaker at 200 revolutions per minute for 20 hours. Each day, double the concentration of the antibiotic from that of the previous day.The next day, transfer 2 microliters of the respective culture to fresh 96 well plate and continue this everyday. Record the optical density reading at 600 nanometers from the individual wells on a plate reader. A critical step of the protocol is the experimental evolution and their increasing concentrations of antibiotics.The rate of change of antibiotic concentrations is one of the parameters that can be modified to promote the evolution of antibiotic resistance. Again, incubate the plate at 37 degrees Celsius on a shaker at 200 revolutions per minute for 20 hours. Then measure the optical density reading at 600 nanometers for each surviving population everyday on the plate reader.Freeze the populations periodically, every three to five days. Extract the total DNA from the evolved populations and the clones from different strains and treatments. Next, measure the absorbance ratios at 260:280, and 260:230 nanometers on the spectrophotometer to determine the quality of the DNA.Then use a fluorescent DNA binding dye and confirm the concentration of the DNA by measuring the fluorescence on the plate reader. Additionally, subject the DNA to agarose gel electrophoresis to confirm that there is no DNA degradation or RNA contamination. Then, perform deep DNA sequencing with samples obtained from whole populations and individual clones to study the genetic basis of antibiotic resistance.This study is an example of the possible outcomes of the experimental system using three e coli strains. These strains are e coli MG1655, MG1655 carrying the resistance gene in the chromosome MG1655 resA and MG1655 carrying the resistance gene in the multicopy plasmid, MG1655 pRESA. The antibiotics used are ceftazidime and meropenem.This left panel shows the survival curves under increasing concentrations of ceftazidime. From the plot, it is clear that the subset of population of strain MG1655 pRESA are able to grow beyond 14 days even after doubling the ceftazidime concentration each day. On the contrary, the other strains do not survive beyond 8 days when exposed to similar concentrations of ceftazidime.However, the same strains do not show significant differences in the survival period when exposed to similar concentration of another antibiotic, meropenem. All the three strains survive only between 6 to 8 days when exposed to meropenem. This indicates that the presence of the gene resA in the multicopy plasmid potentiates the evolution of resistance against ceftazidime but not meropenem.Once mastered, the complete experimental protocol can be performed ina few weeks if done properly. After watching this video, you should have a good understanding of how to investigate the role of multicopy plasmids in the evolution of innovations in bacteria.

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