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How to isolate two plasmids from E. Coli strand?


I have an E. coli cell line I use to express a protein using a two plasmid system. One confers AmpR and one KanR.

For mutagenesis I would like to separate the plasmids to increase efficiency of the mutagenic PCR reaction. I was able to cure the cells of the KanR plasmid because it is low copy whereas the AmpR is high copy so I could easily grow the cells in ampicillin only and I ended up with only the AmpR plasmid. However I was unable to do the same to isolate the KanR plasmid.

My next idea was to run the plasmids through a gel and do an extraction. Since I didn't do a digest it was very hard to differentiate the bands and I ended up getting a negligible yield. I believe the plasmids were simply smeared through the gel due to tertiary structures.

My final hope is to perform a digest on the plasmids using a restriction enzyme that cuts each plasmid once. My concern is that I will not be able to get the stoichiometry correct due to the plasmids both being simulatenously present in vastly different concentrations.

Does anyone have any tips on how else I could isolate the plasmids? Or maybe does anyone have experience running a digest on plasmids with unknown concentrations? I really need help doing this. The mutagenesis project has been going on for months and this isn't even a molecular biology lab… so it's detracting time from main research goals!


A retransformation should get you a pure plasmid. Transform the plasmid mix into empty E.coli (don't use too much DNA), plate on Kan / Amp depending on what you need. The probability that one cell took up both plasmids is extremely low, and you could check with colony PCR just to be sure.

Otherwise: ask around, there must be (old) stocks of the single plasmids around, or glycerol stocks of E.coli containing only one of the plasmids.

Third option: take a look at your mutagenesis method. For your needs there might be techniques around that'll work on a mixture of plasmids. If you just need one variant or a single site saturation library you could use Quikchange for example. I don't see a reason why this wouldn't work on a mix of plasmids.


Plasmid analysis of Escherichia coli isolates from South Korea co-producing NDM-5 and OXA-181 carbapenemases

Recently, Escherichia coli isolates co-producing New Delhi metallo-β-lactamase (NDM)-5 and oxacillinase (OXA)-181 were identified in a tertiary-care hospital of South Korea. Isolate CC1702-1 was collected from urine in January 2017 and isolate CC1706-1 was recovered from a transtracheal aspirate of a hospitalized patient in May 2017. Carbapenemase genes were identified by multiplex PCR and sequencing, and whole genome sequencing was performed subsequently using the PacBio RSII system. Both E. coli isolates belonged to the same clone (ST410) and were resistant to all β-lactams including carbapenems. We obtained whole plasmid sequences of the isolates: pCC1702-NDM-5 from CC1702-1 and pCC1706-NDM-5 and pCC1706-OXA-181 from CC1706-1. The two E. coli isolates belonged to the same clone (ST410) and they were completely resistant to all β-lactams, as well as carbapenems. Two blaNDM-5-harboring plasmids belonged to the same incompatibility group, IncFIA/B, and consisted of 79,613 bp and 111,890 bp with 87 and 130 coding sequences, respectively. The genetic structures of the two blaNDM-5-bearing plasmids, which were distinct from the blaNDM-5-bearing plasmids from the Klebsiella pneumoniae isolates previously transmitted from the United Arab Emirates (UAE) to South Korea, differed from each other. While pCC1702-NDM-5 showed high degree of identity with the plasmid from a multidrug-resistant isolate of Citrobacter fruendii P5571 found in China, pCC1706-NDM-5 was very similar to the plasmid from a multidrug-resistant isolate of E. coli AMA1176 found in Denmark. pCC1706-OXA-181, which was a 51 kb, self-transmissible IncX3 plasmid, was identical to the E. coli plasmids pAMA1167-OXA-181 from Denmark and pOXA-181-WCHEC14828 from China. Plasmids harboring blaNDM-5 in E. coli isolates might not be transferred from K. pneumoniae isolates co-producing NDM-5 and OXA-181. They probably originated from multiple sources.

Keywords: Carbapenemase New Delhi metallo-β-lactamase (NDM)-5 Oxacillinase (OXA)-181.


CRISPR-Cas experiments for schools and the public

The "gene scissors" CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas and a step-by-step tutorial for the practical work. Though the experiment is robust, inexpensive and simple it should be noted that guidance by an expert instructor is required. Based on our experience, a full day lab course has a positive influence on the participants' attitude towards research in general. This is true for high school students as well as non-scientists (age groups 16-70 years).

Keywords: CRISPR Cas9 Class room experiment E. coli Educational module lacZ.

Copyright © 2019 Elsevier Inc. All rights reserved.

Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Figures

Determination of plasmid copy number. A plasmid-containing bacterial strain was grown in a minimal salts medium plus Casamino Acids and glucose. Labeling and lysis were carried out, and the lysate was centrifuged in the CsClethidium bromide gradient. Seven-drop fractions were collected on microtiter trays and assayed for radioactivity.

FIGURE 1

Determination of plasmid copy number. A plasmid-containing bacterial strain was grown in a minimal salts medium plus Casamino Acids and glucose. Labeling and lysis were carried out, and the lysate was centrifuged in the CsClethidium bromide gradient. Seven-drop fractions were collected on microtiter trays and assayed for radioactivity.


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Research output : Contribution to journal › Article › peer-review

T1 - Sequence analysis of two cryptic plasmids from Bifidobactenum longum DJO10A and construction of a shuttle cloning vector

N2 - Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type on consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJHIOS had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coll ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

AB - Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type on consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJHIOS had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coll ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.


Comparative Biology of Two Natural Variants of the IncQ-2 Family Plasmids, pRAS3.1 and pRAS3.2

FIG. 1 . Comparison of the genetic maps of the pRAS3 plasmids with pTF-FC2 and pTC-F14. Percentages below the plasmid backbone genes of pTF-FC2 and pTC-F14 indicate the percent amino acid sequence identity of the gene product with that of the pRAS3 plasmids. Percentages below the oriT and oriV regions indicate nucleotide sequence identity. Plasmids pRAS3.1 and pRAS3.2 have different numbers of 6-bp repeats and 22-bp iterons, while the nucleotide sequence of each repeat or iteron is identical, as indicated below pRAS3. FIG. 2 . The intergenic sequence between the oriT and mobB of pRAS3.1, showing the position of the 6-bp repeats. The oriT is underlined and the imperfect inverted repeat within the oriT is indicated by broken inverted arrows. The conserved hexameric nick site is indicated in bold with a vertical arrow indicating the putative nick position. The 6-bp CCCCCG repeats are labeled 1 to 5. The first repeat consists of only 5 bp, as it lacks a cytosine base. A putative promoter with a near-consensus −35 region and a weak −10 region is shown in bold italics and is separated by a 17-bp spacer. FIG. 3 . Alignment of oriT regions of IncQ-2 plasmids and the IncPα plasmid RP4, showing the sequence divergence that could be tolerated by the Mob proteins of plasmid pRAS3 while they were still able to mobilize DNA from an oriT. A vertical arrow indicates the relaxase nic site at which single-stranded cleavage takes place as determined for plasmid RK2/RP4 (29). FIG. 4 . Phylogenies of toxin-antitoxin proteins of pRAS3 and comparison with closely related proteins as well as the more distantly related PemIK and MazEF proteins. (A) Antitoxins were as follows: Aromatoleum aromatium, CAI08016 Bartonella tribocorum, CAK00897 Dinoroseobacter shibae, YP_001541878 Nitrosomonas europaea ATCC 19718, CAD85218 Xanthomonas axonopolis pv. citri strain 306, NP_644761 Xanthomonas campestris pv. vesicatoria strain 85-10, CAJ19793 Xylella fastidiosa Ann-1, ZP_00682677 E. coli MG1165 MazE, AAA69293 plasmid R100 PemI, P13975. (B) Toxins were as follows: Aromatoleum aromatium, CAI08015 Bartonella tribocorum, CAK00896 Chlorobium ferrooxidans, EAT59633 Nitrosomonas europaea ATCC 19718, CAD85217 Pseudomonas syringae pv. phaseolicola, AAZ37969 Xanthomonas axonopolis pv. citri strain 306, NP_644760 Xanthomonas campestris pv. vesicatoria strain 85-10, CAJ19792 E. coli MG1165 MazF, AAA69292 plasmid R100 PemK, P13976. FIG. 5 . Loss of the low-copy-number test plasmid, pOU82, with and without the PemIK-like TA genes from pRAS3 in the absence of plasmid selection.

Animation 34: Genes can be moved between species.

Stanley Cohen and Herbert Boyer transform bacteria with a recombinant plasmid, and Doug Hanahan studies induced transformation.

Hello, I’m Stanley Cohen… Hello, I’m Stanley Cohen…and I’m Herbert Boyer. In 1972, we were at a biology conference in Hawaii. At the time, I was studying bacterial resistance to antibiotics, and Herbert was studying restriction enzymes. We realized we could work together to recombine genes from different bacteria into one DNA molecule. We used genes from two drug-resistant strains of E. coli bacteria — one gene provides resistance to the antibiotic tetracycline, and the other provides resistance to kanamycin. Each gene is carried on a plasmid in E. coli. Plasmids are small rings of DNA that exist independently of the main bacterial chromosome. They can be replicated and passed on to progeny. I named these plasmids p for plasmid and SC for Stanley Cohen. The plasmid pSC101 carries a gene for tetracycline resistance, and pSC102 carries a gene for kanamycin resistance. We grew the bacterial strains that carried these plasmids, and then we isolated the plasmid DNA. We added the restriction enzyme EcoRI to the plasmid DNA. EcoRI cuts each DNA strand off-center of the recognition site, producing short, single-stranded sequences called "sticky" ends. We mixed the cut plasmids and added DNA ligase. Fragments with EcoRI ends are complementary. This allows fragments to recombine with any other. Hydrogen bonds align two sticky ends, until the ligase repairs the sugar-phosphate bonds to create a stable recombinant molecule. Our objective was to combine the kan r gene and the tet r gene on one plasmid. However, other sorts of molecules were ligated together from the parts. Before we could isolate the recombinant plasmid we wanted, we needed a way to get our ligated plasmids into E. coli. Classic experiments by Oswald Avery and his group showed that Pneumococcus bacteria are "transformed" to virulence when they take up DNA from virulent strains. However, natural transformation is a rare event, so we used a chemical method developed in 1970 by Mandel and Higa at the University of Hawaii. This involved mixing the bacteria and DNA in a suspension of cold calcium chloride at freezing temperature. Then, we rapidly raised and lowered the temperature to create a "heat shock." This technique induces the bacteria to take in plasmid DNA. We spread the transformed bacteria onto a culture plate containing tetracyline and kanamycin. Only transformed bacteria containing both kinds of resistance genes could grow in the presence of both antibiotics. This result was consistent with the bacteria being transformed with a recombined plasmid containing both the tet r and the kan r gene. However, it was also possible that some bacteria had been doubly transformed by religated versions of the original plasmids. Restriction analysis showed that some of the colonies had, indeed, been transformed by a recombinant plasmid. We were able to tell which was which when we cut the plasmids and ran them out on an agarose gel. (Roll over each band to see the difference.) We had made the first recombinant plasmid. Several months later we showed that these same methods could be used to recombine genes from eukaryotic and prokaryotic organisms. We inserted a frog gene into an E. coli plasmid. The resulting bacteria produced frog RNA. Hi, I’m Doug Hanahan. As a graduate student at Harvard, I made the first thorough study of induced transformation of E. coli. Here are my ideas about what happens when bacterial cells are transformed using the Mandel and Higa method. During rapid growth, the cell membrane of E. coli has hundreds of pores, called adhesion zones. The cell membrane itself is made up of lipid molecules that have negatively-charged phosphates. Even though the adhesion zones are physically large enough to admit plasmid DNA, the negatively-charged phosphates on the DNA helix are repelled by those on the lipids. Theoretically, Ca 2+ ions from added calcium chloride can complex with the negative charges, creating an electrostatically neutral situation. Also, lowering the temperature congeals the lipid membrane — stabilizing the negatively-charged phosphates and making them easier to shield. Heat shock creates a temperature imbalance on either side of the bacterial membrane, which may set up a current. With the "ionic shield" in place, the DNA is then swept through the adhesion zone. Techniques like transformation and recombinant DNA have created the field of biotechnology. It is now possible to engineer bacteria to make important human proteins like insulin. However, in order to get bacteria to make insulin or any other eukaryotic protein, a number of factors need to be considered. As you learned in Concept 24, genes in eukaryotic animals have introns — sections of noncoding DNA. Bacteria do not have introns in their genes, and so they do not have the biochemical machinery to remove introns. There is also another consideration. Some eukaryotic proteins are processed after translation. For example, insulin is first translated as preproinsulin, which is 108 amino acids long. The first 24 amino acids are the signal sequence that leads preproinsulin out of the cell. As the protein leaves the cell, the signal sequence is cleaved off, leaving proinsulin, which is stored in the pancreas for further processing. Proinsulin folds into a looped structure and disulfide bridges are made between cysteine amino groups spanning the protein. A 33 amino acid stretch is cleaved off leaving the mature insulin protein. Bacteria cannot process preproinsulin into insulin. So, to get bacteria to make usable insulin, a few tricks were used. First, instead of copying the insulin mRNA, DNA was made based on the protein sequence of the two insulin chains — A and B. Then DNA polymerase was used to make the second strand. These are the double-stranded DNA fragments that are inserted into plasmids. Each DNA fragment is inserted into the -galactosidase gene on a plasmid. The plasmids also have the tetracycline resistance gene. The plasmids are then transformed into bacteria. Tetracycline is added to kill off any untransformed bacteria. The transformed bacteria are grown, then the -galactosidase and insulin fusion protein is harvested and purified. The -galactosidase part of the protein is cleaved off and discarded. Finally, the two protein chains are mixed together. Under the right conditions, the disulfide bonds form and usable human insulin has been made from bacteria.

e coli bacteria, dna ligase, bacterial resistance to antibiotics, plasmid dna, oswald avery, dna molecule, herbert boyer, antibiotic tetracycline, restriction enzymes, hydrogen bonds, bacterial chromosome, dna strand, pneumococcus bacteria, bacterial strains, stanley cohen, restriction enzyme

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TS and ET conceived the study. AL, OS, and TS collected the data. AL and TS analyzed and interpreted the data, and wrote the first draft of manuscript. CB helped to annotate the plasmids. SH and MF performed the sequencing. ET provided thorough critical revisions. All authors contributed to the article and approved the submitted version.

This project was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under agreement no. 2013-67019-21375, the REU program “Molecular and Organismal Evolution” of the National Science Foundation (research grant #1460696), the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #P20GM103408, and by an Undergraduate Research Grant from the Office of Undergraduate Research at the University of Idaho. The genome sequencing was carried out by the IBEST Genomic Resources Core, and made possible thanks to NIH NIGMS (Award No. P30 GM103324). AL was supported in part by the College of Science Undergraduate Research Grant at the University of Idaho.


Conclusions

We developed a novel cloning method that provides an alternative approach to DNA assembly. This method is independent of restriction sites and DpnI treatment, and does not introduce undesired operational sequences at the junctions of functional modules. This new method simplifies complex cloning procedures in which long stretches of DNA can be inserted into circular plasmids in an unrestricted way, and the efficiency does not decrease for long inserts up to 20 kb. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput studies. The protein of interest is expressed without the addition of extra residues originating from the cloning procedure, making it an attractive alternative method for structural genomics.


Transcription Activation (SAM) Vectors

CRISPR/Cas9 Synergistic Activation Mediator (SAM) is a protein complex engineered to enable robust transcriptional activation of endogenous genes – either a single gene at a time, or up to 10 genes simultaneously in the same cell. SAM takes advantage of the specificity and ease of reprogramming of Cas9 nucleases, which are targeted to a specific locus in the endogenous genome by guide RNA. Through a license with the Broad Institute*, GenScript offers validated SAM gRNA sequences to target any coding region in the human genome, as well as complimentary design of SAM gRNA for any other species. SAM guide RNA sequences are custom-synthesized and cloned into efficient lentiviral vectors, and accompanied by the Cas9-VP64 and MS2-P65-HSF1 components that form the three-part SAM complex.