Cloning Should Be Simple: <i>Escherichia coli</i> DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

<div><p>Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex <i>in vitro</i> or <i>in vivo</i> assembly reactions followed by plasmid propagation in recombination-impaired <i>Escherichia coli</i> strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids <i>in vivo</i>. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of <i>E</i>. <i>coli</i> may enable productivity gain across disciplines involving recombinant DNA work.</p></div

pUC19-<i>lacZα</i> assembly assay.

<p>(<b>A</b>) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the <i>lacZα</i> gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene (<i>bla</i>) and the origin of replication. The fragments shared 50-bp homology at both ends. (<b>B</b>) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137466#pone.0137466.s009" target="_blank">S3 Table</a>). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. (<b>C</b>) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.</p

<i>In vivo</i> DNA assembly and cloning in <i>E</i>. <i>coli</i> DH5α.

<p>(<b>A</b>) <i>E</i>. <i>coli</i> DH5α-mediated DNA assembly involves only two basic steps: 1) preparation of fragments with homologous ends and 2) introduction of the fragments into competent cells. This approach minimizes the time and reagents required for DNA assembly in comparison to other common methods, which contain a separate assembly step before the introduction of the constructed plasmid into a recombination-impaired cloning strain such as DH5α (<b>B-D</b>). Assembly is typically carried out either with added enzymes <i>in vitro</i> (<b>B</b>), or <i>in vivo</i>, using as a host <i>S</i>. <i>cerevisiae</i> (<b>C</b>) or specialized <i>E</i>. <i>coli</i> strains expressing the λ Red or RecET phage-based systems (<b>D</b>).</p

Knockout cassette assembly for the deletion of GSU 1371 from <i>Geobacter sulfurreducens</i>.

<p>(<b>A</b>) The GSU 1371 knockout construct was assembled from four fragments including 0.5-kb sequences upstream and downstream of GSU 1371, the kanamycin cassette (amplified from pET28a), and the PCR-linearized pBR322 vector. All adjacent fragments shared 50-bp end homology. In five- and six-fragment assembly experiments, a fifth site (5-F) and both the fifth and sixth sites (5-F and 6-F), respectively, were used to make additional junctions with homology for assembly. (<b>B</b>) Assembly efficiency as a function of DNA concentration was examined using plates containing both Kan and Amp. Colony PCR confirmed the correct insertion in 30/30 transformants tested (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137466#pone.0137466.s004" target="_blank">S4 Fig</a>). (<b>C</b>) The size of the fragments upstream and downstream of GSU 1371 was varied to test the effect on DNA assembly efficiency. Reducing the length of fragments to 250 bp and less resulted in a substantially lower number of colonies. Molar ratio of insert fragments-to-vector was maintained at 5:1 in all experiments.</p

MOESM1 of Two inhibitors of yeast plasma membrane ATPase 1 (ScPma1p): toward the development of novel antifungal therapies

Additional file 1: Table S1. A list of additional antifungal compounds found in our whole-cell screen. Figure S1. IC50 curves for the cell-free, vesicle-based ScPma1p assays. Figure S2. IC50 curves for the whole-cell assays. Figure S3. Compound IC50 values against whole-cell ABC16-Monster yeast, with and without two distinct spiroindolone-binding-pocket ScPMA1 mutations (L290S and P399T)

Assembly of Large, High G+C Bacterial DNA Fragments in Yeast

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with <i>Synechococcus elongatus</i> PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that <i>S. elongatus</i> DNA fragments over ∼200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process

Rapid Chagas Disease Drug Target Discovery Using Directed Evolution in Drug-Sensitive Yeast

Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds’ molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here, we use directed evolution and whole-genome sequencing of a drug-sensitive <i>S. cerevisiae</i> strain to identify the yeast ortholog of <i>Tc</i>Cyp51, lanosterol-14-alpha-demethylase (<i>Tc</i>Cyp51), as the target of MMV001239, a benzamide compound with activity against <i>Trypanosoma cruzi</i>, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another <i>Tc</i>Cyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug–protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other antitrypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for <i>Tc</i>Cyp51 inhibition

Remoted instantaneous frequency measurement system using optical mixing in highly nonlinear fiber

A novel instantaneous frequency measurement using all optical mixing is demonstrated. This system can provide a measurement of RF frequency while requiring no high speed electronic components and remoting all components of the measurement system to the receiver

Malaria Box Heatmap.

<p>Shown are selected data from the HeatMap (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s002" target="_blank">S1 Table</a>) for the 400 Malaria Box compounds. Each column represents an assay (grouped by category), compounds are represented in rows. The red-green gradient represents higher to lower activity. Favorable PK activities are scored green. <i>Pf</i>: <i>Plasmodium falciparum</i>, <i>Pb</i>: <i>Plasmodium berghei</i>, PK: pharmacokinetics, sol.: solubility, hERG: human ether-a-go-go channel inhibition, DDI: drug-drug interactions (predicted).</p

Antiprotozoal Malaria Box compounds with activity in biological assays and lacking toxicity at therapeutic levels.

<p>Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like.</p