Gene Construction Kit Download EXCLUSIVE
Atzin Gendernalik
PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.
Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.
TetR-regulated promoter library data were used in conjunction with in silico modeling to construct negative feedforward loop (NFL) gene networks with different predicted input-output functions. (A) Schematic of the network, where PTEF1 =TEF1 promoter, PLibT = TetR-regulated promoter library, and POR-LT = LacI-TetR dual-regulated promoter. (B) In silico modeling of the network from component properties predicts yEGFP expression (output) in response to varied concentrations of ATc and IPTG (inputs) when three different TetR-regulated promoters are used. (C) The three networks were assembled in S. cerevisiae, and median yEGFP expression was measured by flow cytometry after 22 hours growth of cells in media supplemented with 2% galactose plus varying concentrations of ATc and IPTG. Error bars show the standard deviation of the gated cell population.
The synthetic networks tested in Figure 3B,E,F were used to control the timing of yeast sedimentation caused by flocculation. (A) Schematic of flocculation gene networks. Flocculation is regulated by replacing yEGFP and PLX in the gene network shown in Figure 3A with FLO1 under the control of the L7 promoter (PL7). (B) Rescaled yEGFP data from Figure 3B,E,F were used to project temporal FLO1 expression levels and predict the timing of cell sedimentation due to flocculation (details in Supplementary Information). (C) The timing of sedimentation from the three synthetic networks. Cultures induced by growth with 250 ng/ml ATc for 36 hours were washed twice and grown at high OD600 with shaking and diluted into fresh media every 12 hours, until sedimentation cleared the suspension. Images shown here are 1ml cultures at 12 hour intervals, 10 minutes after brief vortexing. Controls: - = growth in 10 mM IPTG, + = growth in 250 ng/ml ATc.
In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.
The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.
Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression.
Strategies for construction of replacement vectors. On the left, the classical strategy for construction of replacement vectors is shown. It consists of two successive restriction and ligation based cloning steps. On the right the single four fragment USER friendly cloning method is shown. The figures are not drawn to scale. hph = hygromycin phospho-transferase expression cassette (selection marker).
The USER (uracil-specific excision reagent) Friendly cloning technique (New England Biolabs) allows directional cloning of PCR products, independently of restriction enzyme cleavage of the PCR amplicon and DNA ligase for fusion of the amplicon with the vector ends. Instead, vector-specific overhangs (in this paper 9 bp) containing a single 2-deoxyuridine nucleoside, are included in the 5' end of each primer designed to amplify the desired genomic target (Figure 2A). The resulting PCR amplicon (double stranded) is subsequently treated with the USER enzyme mix (Uracil DNA glycosylase and DNA glycosylase-lyase Endo VIII) to create unique 3' single-stranded extensions (Figure 2B). Compatible overhangs (9 bp) in the vector are generated by the combined digestion with a standard restriction enzyme (PacI) and a nicking enzyme (Nt.BbvCI), where the spacing of the respective recognition sites determines the length of the 3' single stranded overhangs (Figure 2C). Annealing of the digested vector and the USER-treated PCR amplicons enables the formation of a stable recombinant molecule that can be used directly in chemical transformation of E. coli without prior ligation (Figure 2D). The DNA pieces are covalently linked by the formation of phosphodiester bonds in vivo, most likely catalyzed by the endogenous E. coli DNA repair system (Figure 2E).
The USER Friendly cloning strategy for single step construction of replacement vectors. A) Amplification of the two homologous recombination sequences (HRS) with primers that contain 5' deoxyuridine extensions. B) Treatment of the PCR amplicons with USER enzyme mix, resulting in the generation of unique 3' single stranded overhangs. The USER enzyme solution is a mixture of Uracil DNA glycosylase and DNA glycosylase-lyase Endo VIII. The Uracil DNA glycosylase recognises the 2'-Deoxyuridine base in the primer portion of the PCR amplicon and excises the uracil nucleobase, resulting in an abasic position [30]. The presence of an abasic site in the DNA permit the DNA glycosylase-lyase Endo VIII to break the phosphodiester backbone at both the 3' and 5' sides of the abasic position, resulting in a single strand break [31]. The resulting short 5' stretch of the original primer then dissociates, leaving the PCR fragment with a 9 bp long 3' single stranded overhang. C) Design of the USER vector for targeted gene replacement in fungi, with two unique USER cloning sites (LB and RB). Each of the UCS's consists of a PacI site (Red), two Nt.BbvCI sites (blue) and two times two unique base pairs (yellow, green, gray and pink) ensuring directional cloning of the inserts. Digestion of the vector results in the generation of two DNA fragments with four unique 9 bp long 3' overhangs. D) Mixing and annealing of the two vector DNA fragments and the two inserts. The four unique 3' overhangs ensures correct annealing between the four DNA fragments. E) Transformation into E. coli, where covalent bonds are formed between the base-paired DNA fragments. F) Screening for correct transformants by colony-PCR using the HRS specific primer pairs that were used in step A. The figures are not drawn to scale.
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