Initially, 86 contigs were obtained with N50 of bp, which were then manually assembled to complete the genome sequence. The completed genome sequence of E.
The assembled genome was then used to identify structural variations of E. Based upon the genomic positions of E. After identifying the structural variations, the SNVs were verified by matching to the assembled E. On top of finding SNVs and structural variations, E. Considering this ratio, it was apparent that the SNVs of E. However, the genomic positions of the SNVs were relatively enriched around the replication origin 3.
Comparison of the mutated genes between E. A SNVs in E. Asterisks and question marks in the amino acid change indicate stop codon and unknown, respectively. D Confirmation of mutations in the identified genes by Sanger sequencing. Mutations in E.
We suggest that the effects of the SNVs in genic regions are beneficial to the phenotype of E. In order to determine whether the mutation was a sequencing error, we performed Sanger sequencing of the SNV regions of both E. In this study, we found novel nucleotide variations of E. To our knowledge, genetic manipulation of phoQ has not been reported in E. We observed SNVs in genes whose products directly interact with the E. These mutations resulted in changes in the amino acid sequence of all of proteins except dadX.
Unfortunately, a very limited number of studies have investigated the protein structures of the mutated genes, and the only available protein structure is that of glmU , whose mutated site is not the active site of the protein Olsen, Vetting and Roderick Unlike these proteins, mipA encodes the MltA-interaction protein, a protein essential for constructing the peptidoglycan layer of the cell envelope in Gram-negative bacteria Vollmer, von Rechenberg and Holtje Interestingly, a periplasmic signal peptide sequence located at the N-terminus of mipA was mutated in the E.
Considering mipA function, which holds murein synthase and murein hydrolase onto a scaffold protein, it is predicted that the lack of a localization of mipA would affect cell membrane composition, which, in turn, may increase plasmid transformation efficiency.
We sequenced the genome of E. In particular, we detected several SNVs in peptidoglycan- and lipid membrane-related genes. Owing to the modifications in the peptidoglycan layer and the outer membrane, mediated by mutations, transformation of external DNA into the cell can be more efficient.
In addition, the laboratory strain may be more resistant to stresses from the environment, like multivalent ions, owing to mutations in the sensor kinase and response regulator. Recent advances in sequencing technologies have facilitated the investigation of mutations in wild-type and laboratory-engineered bacterial cells Conrad, Lewis and Palsson For example, ALE allows one to monitor a strain in a given environment and to measure changes over time by sequencing the adapted genome Cheng et al.
These results would unravel the relationship between the genotype and phenotype, which can be advantageous in developing an engineering host. This suggests that most of the SNVs are derived during the development stage of E. Like E. However, owing to the unexpected random mutations, further investigation of the developed strain is required to understand the exact relationship between the genotype and phenotype of the strain.
Google Scholar. The complete genome sequence of Escherichia coli K Science Engineering Escherichia coli for production of functionalized terpenoids using plant Ps Nat Chem Biol 3 7.
Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol Nat Commun 5 Dhamankar H Prather KL Microbial chemical factories: recent advances in pathway engineering for synthesis of value added chemicals Curr Opin Struct Biol 21 Evolutionary potential, cross-stress behavior and the genetic basis of acquired stress resistance in Escherichia coli Mol Syst Biol 9 Ferenci T The spread of a beneficial mutation in experimental bacterial populations: the influence of the environment and genotype on the fixation of rpoS mutations Heredity Serruto D Galeotti CL The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression Microbiology Structure of the O antigen of Escherichia coli K and the sequence of its rfb gene cluster J Bacteriol Divergence involving global regulatory gene mutations in an Escherichia coli population evolving under phosphate limitation Genome Biol Evol 2 Oxford University Press is a department of the University of Oxford.
It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Yoseb Song , Yoseb Song. Oxford Academic. Bo-Rahm Lee. Suhyung Cho. However, the cloning efficiency was up to times lower than that reported in our experiments. Importantly, the higher cloning efficiencies observed with our method make it possible to assemble more fragments up to six in our experiments and simplify high-throughput cloning due to substantially lower requirements for DNA concentrations.
Based on the experimental descriptions provided in the other study, we believe that several factors may explain the discrepancy. The authors used 30 bp homologous overlaps in most of their experiments, while we designed 40—50 bp overlaps for all of the reported assemblies. The transformation efficiency of the competent cells prepared in-house using rubidium chloride method [ 36 ] was not stated and it is possible that it was substantially lower than that of the commercial cells used in our work.
Finally, the differences in the transformation protocols may also affect the overall cloning efficiency in our methods, although it is likely that the protocols should be optimized for the specific cells used.
In these studies the cloning efficiency is also relatively low and in two of them [ 19 , 20 ] the proposed basis of assembly was attributed to the annealing of complementary single stranded DNA overhangs common to PCR products. However, in the study by Jacobus and Gross [ 21 ], this hypothesis is disproven in cloning experiments using restriction enzyme digested products, which show definitively that the assembly of the DNA fragments occurs in vivo.
Together, our study along with those mentioned above demonstrate the applicability of in vivo cloning in E. In the pUC lacZ experiments, only a few white colonies were observed in both experimental and vector-only transformations, suggesting that non-homologous end joining was extremely rare in the E. It is therefore somewhat surprising that the fidelity of the multi-fragment assembly frequency of colonies containing a correctly assembled plasmid among colonies tested is lower with single antibiotic selection than that observed for single fragment cloning, and the reason for this is not clear.
One possible explanation is that the greater variety of sequences at double-strand breaks increases the chance of having unintended end-microhomologies that may be able to recombine [ 29 ], forming unwanted products. Plasmid DNA is commonly transformed into E. We hypothesize that the observed difference in assembly efficiency can be explained by the different mechanisms of DNA transport in the two approaches. The conditions employed for the transformation of chemically competent cells are believed to promote the formation of multiple channels per cell as well as DNA crowding at the cell membranes.
On the other hand, in electroporation-induced transformation, the DNA appears to enter the cells in a stochastic manner through the pores formed during the short electrical pulse. Thus, the probability that the same viable competent cell will take up all DNA fragments required for the correct assembly would decrease exponentially with each additional fragment. Based on the observations of Koskela and Frey, [ 30 ] it is also possible that in the transformation method of chemically-induced cells, DNA fragments to be assembled begin to interact during the incubation of DNA with competent cells before heat shock, further enhancing the efficiency of this method in comparison to electroporation-induced transformation.
It is not clear whether a similar comparison between highly competent chemically induced cells and electroporation induced cells has been performed in the published experiments. Many studies have focused on mechanisms of homologous recombination in E. RecA is the major bacterial recombination protein, essential for repair and maintenance of DNA in the cell [ 39 ].
This was later supported by Lovett et al. The Red system has been studied and employed mainly for engineering of the E. Other mechanisms of RecA-independent recombination have been identified in E.
The lower recombination efficiency thus becomes an advantage, as it allows the same cell to be used to assemble, clone, and amplify the recombinant DNA. As with any method, E. Based on the early Bubeck et al. It is also known that transformation efficiency decreases with size [ 31 ], and it is likely that assembly of fragments larger than 20—30 kb will be challenging.
Furthermore, our results suggest that assembly of more than five fragments is difficult and fidelity decreases with increasing number of fragments. Despite the stated limitations, the method described here should be pertinent to most biological applications that require recombinant DNA.
Its power is in its simplicity, as it reduces cloning to two basic steps of DNA preparation and transformation of commercially available E. It can be readily integrated into a wide range of experimental workflows. Blue colonies indicate correct assembly of the pUC19 construct.
Shown is a plate from an experiment testing the effect of DNA quantity on transformation efficiency, quantified as the number of blue colonies see Fig 2 and S3 Table. Fragments shared 50 bp of homology at their ends see Materials and Methods. A range of vector DNA concentrations was tested while maintaining the insert-to-vector ratio at Thirty colonies were tested using primers 5-F and 5-R S2 Table.
Expected band size was 1. Expected band size was 2. We thank T. Hanly, H. Smith, B. Karas, K. Wise, P. Weyman, R.
Richter, J. Linger, and D. Wilson for helpful discussion. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. PLoS One. Published online Sep 8. Otwell , 2 Ruth E. Anne E. Ruth E. Mark Isalan, Editor. Author information Article notes Copyright and License information Disclaimer. Received Jun 10; Accepted Aug This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
This article has been cited by other articles in PMC. S2 Fig: Assembly of Aspergillus niger cbhA into a custom vector.
S1 Table: High-throughput cloning of cellulase and other carbohydrate-active enzyme genes. S4 Table: In vivo assembly of the knockout cassette for gene in Geobacter sulfurreducens from three fragments into a pBR vector. S5 Table: Comparison of single and double selections on colony number and the fidelity of the GSU knockout construct.
S7 Table: Effect of fragment number on the assembly of a pBRbased knockout construct for the deletion of gene GSU in Geobacter sulfurreducens. Abstract Numerous DNA assembly technologies exist for generating plasmids for biological studies.
Introduction Recombinant DNA technologies have been critical for driving biotechnological advances and facilitating studies aimed at understanding basic biological principles. Open in a separate window. Fig 1. In vivo DNA assembly and cloning in E. Transformation of E. Results Single-fragment cloning If DNA constructs can be generated and propagated in a single organism, the overall workflow in molecular biology can be considerably simplified with far-reaching impacts on scientific advances.
Table 1 Assembly of pUC19 by different commercial strains. Fig 2. Multi-fragment cloning In many instances, it is necessary to assemble several DNA fragments, such as in the construction of fusion proteins, gene knockout cassettes, and large genes from smaller synthesized fragments. Fig 3. Knockout cassette assembly for the deletion of GSU from Geobacter sulfurreducens. Plasmid alteration via self-closure Along with the assembly of one or more fragments into a vector, it is sometimes useful to alter existing plasmids.
Discussion Reducing the number of steps in DNA assembly has several important advantages. PDF Click here for additional data file. S2 Fig Assembly of Aspergillus niger cbhA into a custom vector. S1 Table High-throughput cloning of cellulase and other carbohydrate-active enzyme genes.
S4 Table In vivo assembly of the knockout cassette for gene in Geobacter sulfurreducens from three fragments into a pBR vector. S5 Table Comparison of single and double selections on colony number and the fidelity of the GSU knockout construct.
Acknowledgments We thank T. Data Availability All relevant data are within the paper and its Supporting Information files. References 1. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Nucleic Acids Res. Quan J, Tian J.
Circular polymerase extension cloning of complex gene libraries and pathways. Nat Methods. Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl.
Look for the TR mark in the catalog number for trial size packs. DH5-alpha Chemically Competent E. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt. Quality Control Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below.
Untransformed cells are tested for appropriate antibiotic sensitivity. Procedure for transformation through heat shock utilizing DH5-alpha chemically competent E. Guide to GoldBio's competent cell collection containing transformation efficiencies, characteristics and applications for each of our competent cell lines. Chart containing genotypes and transformation efficiencies for GoldBio's competent cell collection.
This guide provides nomenclature information relating to genotypes and genetic markers of E. Leave Product Feedback. General Guidelines Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Thaw competent cells on ice, and transform cells immediately following thawing.
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