scholarly journals Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

2014 ◽  
Vol 10 ◽  
pp. 2348-2360 ◽  
Author(s):  
Kristen K Merritt ◽  
Kevin M Bradley ◽  
Daniel Hutter ◽  
Mariko F Matsuura ◽  
Diane J Rowold ◽  
...  
Keyword(s):  
Solid Phase ◽  
Pcr Amplification ◽  
Kanamycin Resistance ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Synthetic Dna ◽  
Information Density ◽  
Letter Alphabet ◽  
New Strategy ◽  
Autonomous Assembly

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

scholarly journals Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation

1999 ◽  
Vol 65 (4) ◽  
pp. 1675-1680 ◽  
Author(s):  
Ruben G. Kok ◽  
David M. Young ◽  
L. Nicholas Ornston
Keyword(s):  
Homologous Recombination ◽  
Pseudomonas Putida ◽  
Natural Transformation ◽  
Phenotypic Expression ◽  
Point Mutations ◽  
Pcr Amplification ◽  
Kanamycin Resistance ◽  
Multiple Cloning Site ◽  
Dna Fragments ◽  
Resistance Marker

ABSTRACT Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome ofAcinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacterchromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylEgene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.

A new strategy for total solid-phase synthesis of polymyxins

2015 ◽  
Vol 56 (33) ◽  
pp. 4796-4799 ◽  
Author(s):  
Wei-Liang Xu ◽  
A-Long Cui ◽  
Xin-Xin Hu ◽  
Xue-Fu You ◽  
Zhuo-Rong Li ◽  
...  
Keyword(s):  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Total Solid ◽  
Phase Synthesis ◽  
New Strategy

A New Strategy for Solid-Phase Depsipeptide Synthesis Using Recoverable Building Blocks

2005 ◽  
Vol 7 (4) ◽  
pp. 597-600 ◽  
Author(s):  
Fernando Albericio ◽  
Klaus Burger ◽  
Javier Ruíz-Rodríguez ◽  
Jan Spengler
Keyword(s):  
Solid Phase ◽  
Building Blocks ◽  
New Strategy

scholarly journals Micro Salting-Out Assisted Matrix Solid-Phase Dispersion: A Simple and Fast Sample Preparation Method for the Analysis of Bisphenol Contaminants in Bee Pollen

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2350
Author(s):  
Jianing Zhang ◽  
Fengjie Yu ◽  
Yunmin Tao ◽  
Chunping Du ◽  
Wenchao Yang ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Preparation Method ◽  
Solid Phase ◽  
Sample Preparation Method ◽  
Salting Out ◽  
Bee Pollen ◽  
Matrix Solid Phase Dispersion ◽  
Phase Dispersion ◽  
New Strategy ◽  
Semi Solid

In the present work, a novel sample preparation method, micro salting-out assisted matrix solid-phase dispersion (μ-SOA-MSPD), was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) contaminants in bee pollen. The proposed method was designed to combine two classical sample preparation methodologies, matrix solid-phase dispersion (MSPD) and homogenous liquid-liquid extraction (HLLE), to simplify and speed-up the preparation process. Parameters of μ-SOA-MSPD were systematically investigated, and results indicated the significant effect of salt and ACN-H2O extractant on the signal response of analytes. In addition, excellent clean-up ability in removing matrix components was observed when primary secondary amine (PSA) sorbent was introduced into the blending operation. The developed method was fully validated, and the limits of detection for BPA and BPB were 20 μg/kg and 30 μg/kg, respectively. Average recoveries and precisions were ranged from 83.03% to 94.64% and 1.76% to 5.45%, respectively. This is the first report on the analysis of bisphenol contaminants in bee pollen sample, and also on the combination of MSPD and HLLE. The present method might provide a new strategy for simple and fast sample preparation of solid and semi-solid samples.

scholarly journals Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Matsuzaki ◽  
Eiichi Okamura ◽  
Daichi Kuramochi ◽  
Aki Ushiki ◽  
Katsuhiko Hirakawa ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Genomic Imprinting ◽  
Dna Fragments ◽  
Cis Elements ◽  
Synthetic Dna

Validating Soot Models in LES of Turbulent Flames: The Contribution of Soot Subgrid Intermittency Model to The Prediction of Soot Production in an Aero-Engine Model Combustor

2021 ◽  
Author(s):  
Lívia Pereira Tardelli ◽  
Nasser Darabiha ◽  
Denis Veynante ◽  
Benedetta Franzelli
Keyword(s):  
Gas Phase ◽  
Reference Model ◽  
Solid Phase ◽  
Phase Evolution ◽  
Turbulent Flames ◽  
Engine Model ◽  
Aero Engine ◽  
New Strategy ◽  
Parametric Studies ◽  
Soot Model

Abstract Predicting soot production in industrial systems using an LES approach represents a great challenge. Besides the complexity in modeling the multi-scale physicochemical soot processes and their interaction with turbulence, the validation of newly developed models is critical under turbulent conditions. This work illustrates the difficulties in evaluating model performances specific to soot prediction in turbulent flames by considering soot production in an aero-engine combustor. It is proven that soot production occurs only for scarce local gaseous conditions. Therefore, to obtain a statistical representation of such rare soot events, massive CPU resources would be required. For this reason, evaluating soot model performances based on parametric studies, i.e., multiple simulations, as classically done for purely gaseous flames, is CPU high-demanding for sooting flames. Then, a new strategy to investigate modeling impact on the solid phase is proposed. It is based on a unique simulation, where the set of equations describing the solid phase are duplicated. One set accounts for the reference model, while the other set is treated with the model under the scope. Assuming neglected solid phase retro-coupling on the gas phase, the soot scalars from both sets experience the same unique temporal and spatial gas phase evolution isolating the soot model effects from the uncertainties on gaseous models and numerical sensitivities. Finally, the strategy capability is proven by investigating the contribution of the soot subgrid intermittency model to the prediction of soot production in the DLR burner.

scholarly journals Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

2001 ◽  
Vol 45 (5) ◽  
pp. 1343-1348 ◽  
Author(s):  
Hisakazu Yano ◽  
Akio Kuga ◽  
Ryoichi Okamoto ◽  
Hidero Kitasato ◽  
Toshimitsu Kobayashi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Antimicrobial Agents ◽  
Pcr Amplification ◽  
Penicillin G ◽  
Gene Encoding ◽  
Mature Enzyme ◽  
Dna Fragment ◽  
Lactamase Gene ◽  
Reduced Activity ◽  
Northern Japan

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries bla IMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The k cat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

Sign in / Sign up

Export Citation Format

Share Document