Plasmids are extrachromosomal elements that are ubiquitous in most bacterial species and habitats where they are very important agents of lateral gene transfer as they can migrate between cells and between populations.One of the most striking and prominent examples of such transfer is the dissemination of antibiotic resistance genes that are encoded on plasmids.To build a model plasmid that encodes an antibiotic resistance gene, choose a plasmid backbone and PCR amplify the backbone using a high fidelity polymerase and primers that bind on the plasmid template.In our case we used a pBBR1 plasmid backbone.Next, amplify the resistance gene nptII including the native Tn5 promoter using a high fidelity polymerase.Design the primers for the resistance gene with approximately 20 base pairs of sequence complementarity to the plasmid backbone.After the PCRs, fuse the homology region of the purified resistance gene PCR product to the purified plasmid backbone using isothermal assembly at 50 degrees for 60 minutes.Transform the fused product into E.Coli DH5-Alpha using electroporation.Plate 100 microliter of the mixture on selective media and incubate at 37 degrees for about 24 hours.The plasmid can be then extracted, validated and transformed into the E.coli wild type strain MG1655.This yields strain MG1655 pCON.During antibiotic exposure, bacteria need the plasmid to survive.Thus, the plasmid is under positive selection.Under non-selective conditions, so without antibiotics, the plasmid is disposable to the cell.The aim of our study was to follow the plasmids under such non-selective conditions over time.For this purpose, we equipped the small plasmids with an antibiotic resistance gene and introduced it to Escherichia coli.We conducted an evolutionary experiment and monitored the plasmids frequency via replica plating.The plasmid carrying strain mg1655 pCON is now introduced to an evolution experiment.First, plate mg1655 pCON on LB agar plates supplemented with antibiotics and incubate overnight at 37 degrees.From this plate randomly choose the 8 ancestor colonies and incubate overnight at 37 degrees with constant shaking.At the next day, the populations are transferred to the experiment that is conducted in 96 deep well plates.It is highly important to randomly distribute the replicates across the plate.For example, in a checkerboard approach.In our experiment the cultures are diluted and transferred with different dilution rates and in two temperatures.During every transfer event the dilution treatment is applied and the serial transfer is repeated over a total of 98 transfers.Along the evolution experiment the frequency of plasmids in the population is estimated from the proportion of plasmid carrying cells.Replica plating was popularized by Esther and Joshua Lederberg in the 1950s where they used it in order to study drug resistance in bacteria.The method will enable them to upscale their scanning for phenotypes and at the end of study revealed that penicillin resistance can arise in bacteria due to spontaneous mutations in the genome.To determine the plasmid frequency in the population during the experiment, stationary cultures are serially diluted and plated on non-selective LB agar plates.Adjust the dilution according to a yield of 250 to 500 colonies per plate.The plated populations are incubated for overnight growth at 37 degrees for less than 24 hours.Colonies are best replicated when they're still small.After overnight growth count all colonies using an automated colony counter of the reader's choice.This yields the total bacteria population size in the cultures.Note that colonies at the edge of the plate should be left out.To replicate the plates, you need selective agar plates supplemented with antibiotics, a round block, a metal ring that fits the block, and sterile velvet cloth.The cloth needs to be 100%cotton as this can be sterilized by autoclavation.Place the cloth on the block and fix it with the metal ring.Carefully place the plate with the counted colonies on the fixed velvet cloth surface.Tap the plate in circle movements so that all the plate surface touches the cloth.It is very important that all colonies touch the velvet.Remove the LB plate and place the selective plate on the velvet.Repeat the careful tapping.It is again important that the plate touches the velvet completely.Afterwards, remove the selective plate.The plates are incubated at room temperature overnight.At the next day, both the LB and the antibiotics plate are needed for evaluation.Place the plates one above each other and compare the growth.You can easily spot colony-free spaces.These are the colonies that are not resistant to the antibiotics.Thus, these colonies lost the plasmid.In the end, mark how many colonies are missing on the antibiotics plate.This number gives you the plasmid loss.Repeat the replica plating of all populations during the evolution experiment weekly.Plasmid loss can be caused by plasmid multi malformation.When working on a plasmid construction say for the expression of a given gene in your preferred host strain the native behavior of a plasmid in vivo with regards to the confirmation a state can take is usually something that is overlooked.But in an evolutionary context such confirmation of changes might be of very great importance.The analysis of plasmid confirmations can be a very tricky endeavor.Especially plasmid multimers are difficult to analyze because they can't be identified properly by PCR or by sequencing.On the other side, plasmid multimers are difficult to analyze by gel electrophoresis because of their potentially large size and their slow electrophoretic mobility.Our protocol offers a simple and straightforward method for the screening and identification of plasmid multimers in I would say any given plasmid preparation.To visual plasmid molecules extract the plasmid from 5 mL stationary overnight cell culture using alkaline lysis.Plasmid extraction of low copy plasmids often leads to contamination with host chromosomal DNA that needs to be digested prior to visualization.To remove chromosomal DNA contamination treat the extracted plasmid DNA with Plasmid-Safe
