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We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems. The range of available copy numbers for cloning vectors is largely restricted to the handful of ORIs that have been isolated from plasmids found in nature. Here the authors introduce a plasmid system that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell.

Abstract Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out. EasyClone genetical toolbox allows faster development of yeast strains for biotechnological applications.

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Objective Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant loxP sites present. Results An oligonucleotide containing a mutant loxP 2272 site and a Xho I restriction enzyme sequence was designed and inserted at the Sfi I restriction site located approximately 200 basepairs upstream of the lacZ gene in pBeloBAC11. Clones containing the desired insert were identified by Xho I restriction digests since an additional band was generated. This transposon-mediated deletion technology allows researchers to identify the boundaries of cis -acting elements and genes.

Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Salmonella cerevisiae and Bacillus subtilis . In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

The effect of the addition of sequential C‐terminal tryptophan residues on the fluorescence intensity of GFP was investigated. Tandem repeats of six tryptophan residues markedly decreased fluorescence intensity. This phenomenon is likely to occur because of the inhibition of GFP folding, resulting in insolubility. Exploiting this phenomenon, we constructed a cloning vector that facilitates the identification of recombinant colonies of Escherichia coli by the activation of GFP. Tandem repeats of six tryptophan residues at the C terminus of green fluorescent protein (GFP) markedly decreased the fluorescence intensity. This phenomenon is likely to occur because of the inhibition of GFP folding, resulting in insolubility. Exploiting this phenomenon, we constructed a cloning vector that facilitates the identification of recombinant colonies of Escherichia coli by the activation of GFP.

Transposon mini-Tn7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris. The transposition of the mini-Tn7 into the bacterial genome was detected at a Tn7 attachment (attTn7) site located downstream of glmS1. Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn7 insertion mutant was created. Mini-Tn7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn7 and FLP-FRT systems also work well in Xanthomonas oryzae pv. oryzae.

The genetic modification of lactic acid bacteria being used in medicine and food industries has been limited due to the scarcity of food-grade cloning vectors for the bacteria. The 4.46-kb food-grade cloning vector pUBU constructed in this study consisted of 3 major components from food-approved organisms, the theta-type replicon from pUCL287 of Tetragenococcus halophilus, the lactococcal cadmium resistance (Cdʳ) determinant from pND918 and the promoter of L-lactate dehydrogenase (ldhL) gene from Lactobacillus plantarum. The Cdʳ determinant was used as a dominant selectable marker and the ldhL promoter, a strong constitutive promoter, was used to drive the expression of inserted genes. The newly constructed vector was able to transform several genera of lactic acid bacteria and stable in the bacteria under non-selective pressure for at least 100 generations. In addition, it allowed inserted genes to express in lactic acid bacteria under the control of ldhL promoter. The host range of pUBU extended to Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Tetragenococcus. These results suggest that pUBU is a potential food-grade cloning vector for genetic modification of a wide range of lactic acid bacteria.