The phage T4 DNA ligase in vivo improves the survival-coupled bacterial mutagenesis

Wang, Junshu; Liu, Fapeng; Su, Tianyuan; Chang, Yen‐Chiang; Guo, Qi; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

doi:10.1186/s12934-019-1160-7

Cited by 7 publications

(4 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, our previous work proved that overexpression of the T4 DNA ligase derived from T4 phage in E. coli can significantly promote the repair of intracellular DNA double-strand breaks (DSBs), suggesting that the activity of DNA ligase is of great importance for in vivo DNA assembly. , …”

Section: Introductionmentioning

confidence: 99%

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

Pang

Han

et al. 2022

ACS Synth. Biol.

Self Cite

View full text Add to dashboard Cite

The assembly of DNA fragments is extremely important for molecular biology. Increasing numbers of studies have focused on streamlining the laborious and costly protocols via in vivo DNA assembly. However, the existing methods were mainly developed for Escherichia coli or Saccharomyces cerevisiae, whereas there are few direct in vivo DNA assembly methods for other microorganisms. The use of shuttle vectors and tedious plasmid extraction and transformation procedures make DNA cloning in other microorganisms laborious and inefficient, especially for DNA library construction. In this study, we developed a "phage enzymeassisted in vivo DNA assembly" (PEDA) method via combinatorial expression of T5 exonuclease and T4 DNA ligase. PEDA facilitated the in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and it is applicable to multiple microorganisms, such as Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. The cloning efficiency of optimized PEDA is much higher than that of the existing in vivo DNA assembly methods and comparable to that of in vitro DNA assembly, making it extremely suitable for DNA library cloning. Collectively, PEDA will boost the application of in vivo DNA assembly in various microorganisms.

show abstract

Section: Introductionmentioning

confidence: 99%

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

Pang

Han

et al. 2022

ACS Synth. Biol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…For example, mutagenesis and genetic engineering, as well as adaptive evolution, are the main approaches to improve the performance of strains to build large libraries of mutants . Moreover, the atmospheric and room temperature plasma (ARTP) is a novel mutagenesis technology widely used for microbial strain improvement. − However, the rapid and efficient acquisition of target strains from a large strain variant library through high-throughput screening technology remains a great challenge. The main bottleneck technology is low efficient high-throughput analytical methods during the DBTL cycle.…”

Section: Introductionmentioning

confidence: 99%

An Efficient High-Throughput Screening of High Gentamicin-Producing Mutants Based on Titer Determination Using an Integrated Computer-Aided Vision Technology and Machine Learning

Zhu

Mohsin

et al. 2022

Anal. Chem.

View full text Add to dashboard Cite

The “design–build–test–learn” (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R 2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.

show abstract

“…The T4 DNA structure remains condensed after the structural RNA and protein components have been removed (Hamilton and Pettijohn 1976). T4 DNA ligase is an enzyme that is widely used in molecular biology to repair chromosomal DNA during mutagenesis (Wang et al 2019). The distribution of covalent modifications in T4 DNA is inhibited by singlemolecule DNA sequencing and enzymatic probing (Bryson et al 2015).…”

Section: Introductionmentioning

confidence: 99%

“…T4 DNA ligase is an enzyme that is widely used in molecular biology to repair chromosomal DNA during mutagenesis (Wang et al. 2019 ). The distribution of covalent modifications in T4 DNA is inhibited by single-molecule DNA sequencing and enzymatic probing (Bryson et al.…”

Section: Introductionmentioning

confidence: 99%

Direct visualization of local activities of long DNA strands via image–time correlation

Kang

Sadakane

2021

Eur Biophys J

View full text Add to dashboard Cite

Bacteriophages with long DNA genomes are of interest due to their diverse mutations dependent on environmental factors. By lowering the ionic strength of a hydrophobic (PPh4Cl) antagonistic salt (at 1 mM), single long T4 DNA strand fluctuations were clearly observed, while condensed states of T4 DNA globules were formed above 5–10 mM salt. These long DNA strands were treated with fluorescently labeled probes, for which photo bleaching is often unavoidable over a short time of measurement. In addition, long (few tens of $$\upmu m$$ μ m ) length scales are required to have larger fields of view for better sampling, with shorter temporal resolutions. Thus, an optimization between length and time is crucial to obtain useful information. To facilitate the challenge of detecting large biomacromolecules, we here introduce an effective method of live image data analysis for direct visualization and quantification of local thermal fluctuations. The motions of various conformations for the motile long DNA strands were examined for the single- and multi-T4 DNA strands. We find that the unique correlation functions exhibit a relatively high-frequency oscillatory behavior superimposed on the overall slower decay of the correlation function with a splitting of amplitudes deriving from local activities of the long DNA strands. This work shows not only the usefulness of an image–time correlation for analyzing large biomacromolecules, but also provides insight into the effects of a hydrophobic antagonistic salt on active T4 bacteriophage long DNA strands, including thermal translocations in their electrostatic interactions.

show abstract

The phage T4 DNA ligase in vivo improves the survival-coupled bacterial mutagenesis

Cited by 7 publications

References 39 publications

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

An Efficient High-Throughput Screening of High Gentamicin-Producing Mutants Based on Titer Determination Using an Integrated Computer-Aided Vision Technology and Machine Learning

Direct visualization of local activities of long DNA strands via image–time correlation

Contact Info

Product

Resources

About