Cloning Protocol Research Articles

LyGo: A Platform for Rapid Screening of Lytic Polysaccharide Monooxygenase Production.

Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with 14 different expression vectors in a simple 15 min reaction, thus enabling rapid exploration of several gene contexts, hosts, and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration of its utility, we explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.

ACC: Aerial Cell Cloning protocol for efficient session continuity in LAP replacement for 6G

Abstract Low Altitude Platform (LAP) based aerial cells improve the performance of the cellular network dynamically. However, the small form factor of LAPs constraints the battery size, limiting its hovering time. LAPs should be replaced when the available power or available capacity goes below a threshold, in addition to reasons like a mechanical malfunction. Replacement requires data sessions from all the connected user equipment (UEs) to transfer from the old LAP to the new LAP. Classical schemes like (i) UE handover and (ii) carrier link management support UE mobility and load balancing. The classical schemes have higher latencies, which scales if there are many connected UEs, leading to increased data session interruption if applied in LAP replacement. The interruption leads to a degraded user experience during gaming and multimedia consumption. In this paper, to the best of our knowledge for the first time, we propose a novel Aerial Cell Cloning (ACC) protocol for network management applied during LAP replacement. Through call flow analysis and mathematical modeling backed with extensive system-level simulation, we show that the proposed ACC protocol reduces the session transfer latency by over 68% and brings down the LAP replacement time to a negligible value (99% reduction) when compared to existing classical schemes.

Equity Is Key to Build Back Better after COVID-19: Prioritize Noncommunicable Diseases and Kidney Health.

<h3>ABSTRACT</h3> Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA secondary structure and nucleotide modifications can both interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We find that OTTR robustly captures full-length tRNAs in budding yeast and in mouse testis, with relatively low levels of premature termination at known barriers – 1-methylguanine, N2,N2-dimethylguanine, and 1-methyladenine – to typical reverse transcriptases. Moreover, these and several other nucleotide modifications leave misincorporation signatures in OTTR datasets which enables their detection without any additional protocol steps. Turning to analysis of small RNAs such as tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to “ground truth” Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRFs – including both 5’ and 3’ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly

BackgroundThe ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes.ResultsWe developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated.ConclusionsOur protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Global DNA methylation: role, status and genome-wide approaches to study epigenetic mark in cloned embryos

Somatic cell nuclear transfer (SCNT) technique has been proving its worth for more than two decades now as over 20 different species have been successfully cloned. SCNT protocol for cloning is well established but efficiency in terms of live birth rate is still low. Epigenetic abnormality following nuclear reprogramming is considered as the main culprit behind its low efficiency. DNA methylation is one of the most important epigenetic modifications that directly or indirectly regulate gene expression pattern, development and genome stability. Embryos produced through SCNT are found to express abnormal DNA methylation profile in comparison with in vivo or in vitro produced embryos. In order to improve DNA methylation profile in cloned embryos, a complete database of whole genome is required to find out specific faulty targets. Many techniques including low throughput and high throughput approach has been used to profile DNA methylation pattern in bovine embryos throughout the developmental stages. In the present review, we have compiled the overall status of global DNA methylation, the effect of aberrant DNA methylation on development and evolution in methodologies used for profiling global DNA methylome in cloned embryos.

A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.

Limitations imposed by complementarity

Complementarity is one of the main features of quantum physics that radically departs from classical notions. Here we consider the limitations that this principle imposes due to the unpredictability of measurement outcomes of incompatible observables. For two-level systems, it is shown that any preparation violating complementarity enables the preparation of a non-signalling box violating Tsirelson's bound. Moreover, these "beyond-quantum" objects could be used to distinguish a plethora of non-orthogonal quantum states and hence enable improved cloning protocols. For higher-dimensional systems the main ideas are sketched.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites

BackgroundLigation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage.ResultsHere we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations.ConclusionsMerging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.

Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.

A T-cell reporter platform for high-throughput and reliable investigation of TCR function and biology.

ObjectiveTransgenic re‐expression enables unbiased investigation of T‐cell receptor (TCR)‐intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large‐scale transgenic TCR re‐expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re‐expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat‐based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read‐out of TCR functionality and facilitates high‐throughput screening approaches.MethodsAs a proof‐of‐concept, we transgenically re‐expressed 59 human cytomegalovirus‐specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells.ResultsWe demonstrate that the TPRKO cell line facilitates antigen‐HLA specificity screening via sensitive peptide‐MHC‐multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co‐receptor.ConclusionOverall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high‐throughput investigation of TCR biology.

Cloning and Heterologous Expression of Protein-Coding Sequences in Escherichia coli.

Recombinant protein expression is widely used to produce large quantities of protein for diverse uses including functional characterization of selected sequences and vaccination trials. In the postgenomic era, high-throughput techniques that allow us to manipulate several sequences are needed. Cloning by in vivo recombination is a technique that consists in the insertion of a linear DNA into a linearized plasmid DNA by in vivo recombination using a recA+ E. coli strain. This methodology provides high-throughput cloning with high efficiency without the need for restriction enzyme digestion. In this chapter, we describe two protocols for DNA cloning: one using in vivo recombination and the other by using restriction enzymes. We also describe the application of different conditions to produce functional proteins that needs the incorporation of the amino acid selenocysteine (Sec), like thioredoxin-glutathione reductase enzyme.

Using Lactococcus lactis as Surrogate Organism to Study Group A Streptococcus Surface Proteins.

The isolation of a single Group A Streptococcus (GAS) virulence determinant in functional investigations is challenging, as GAS employs a multitude of virulence factors. The redundancy between many surface proteins such as adhesins also adds complexity and difficulty. Lactococcus lactis is a non-pathogenic Gram-positive species related to GAS that can be an ideal surrogate organism to circumvent this problem. Genetic manipulation in L. lactis is easy, and the mechanisms for processing and cell wall-anchoring of surface proteins are similar to GAS. Lactococci have been extensively used to express heterologous surface proteins from other bacterial species, and modern molecular cloning tools and protocols have been developed. This chapter describes the workflow of generating recombinant L. lactis strains expressing GAS surface proteins and the validation and quantification of their surface expression.

Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes

Providing an improved protocol for cloning of Picea abies (L.) H. Karst. mature trees via Agrobacterium rhizogenes. In reforestation programs, rejuvenation of superior genotypes is of great importance in helping juvenile rootstock production. In this regard, the use of plant tissue culture to produce axillary and/or adventitious shoots from explants of mature trees has been the objective of many researchers. There are a few reports about the successful in vitro rooting of the regenerated shoots from explants of mature trees. There is a very low rate of natural rooting in conifers, between 1 and 5%. Furthermore, they are still hard to root even when auxins are applied. In this study, in vitro propagation of Norway spruce explants and its rooting improvement by A. rhizogenes with the aim of receiving an efficient clonal propagation method was investigated. The effects of BA (0.5, 1, 1.5, and 2 μM) and TDZ (0.0025, 0.025, 0.25, and 2.5 μM) were examined on axillary shoot development from shoot-tip explants of the Norway spruce. The effects of three strains of Agrobacterium rhizogenes (K599, AR15834 and GMI9534) were also examined on enhancement of the root induction. According to the results, after the treatment of 1.5 μM BA, 87.5% of shoot-tip explants produced axillary shoots (the highest percentage among treatments), and also the maximum mean number of regenerated shoots per explant (6.5) was achieved in this treatment. Furthermore, the induction of rooting was performed using IBA (7.5 µM) and IAA (0.5 or 1 μM) in the WPM medium along with keeping the explants in the darkness at 21 °C, which yielded 37.5% and 12.5% rooting, respectively. Moreover, the application of coumarin along with IAA caused an increase of 22% in rooting, compared to the use of IAA alone. Two strains, K599 and AR15834, succeeded in improving the percentage of rooting, with the highest being 54.2% as a result of using the K599 strain alongside 7.5 μM IBA. The proposed treatment can be used efficiently for the propagation of Norway spruce in landscaping and/or reforestation programs.

Rapid Identification of Protein-Protein Interactions in Plants.

Enzyme-enzyme interactions can be discovered by affinity purification mass spectrometry (AP-MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP-MS. The success of AP-MS depends on the levels and stability of the bait protein, the stability of the protein-protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in-gel-digestion AP-MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP-MS experiment constitutes a single sample for LC-MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold-change abundance (FC-A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co-sublocalization of "bait" and "prey," we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC-A values of at least four within-biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme-enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In-Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On-bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control.

Engineering a bioluminescent bioreporter from an environmentally sourced mercury-resistant Enterobacter cloacae strain for the detection of bioavailable mercury.

Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild-type bacterial host derived from a soil environment. In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux , detected mercury (HgCl2 ) at a minimum concentration of 0·2µgl-1 with a linear response profile being maintained between a range of 0·4-1600µgl-1 (R2 =0·9604) with a peak bioluminescent response occurring within 1h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2 =0·9623). The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2µgl-1 ). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination. This study provides new insight into the recruitment of mercury-tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future.

Engineering proteins for allosteric control by light or ligands.

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.

A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products

An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes. Here, I describe the development of three vectors for TA cloning and blunt-end cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR cloning protocol with non-digested circular vectors and PCR products. This direct PCR cloning protocol yielded colony-formation rates and cloning efficiencies that are comparable with those obtained by conventional PCR cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene (https://www.addgene.org/).