A variety of simple and ultra-low-cost methods preparing SLiCE extracts and their application to DNA cloning

Guo, Ruiyan; Zhao, Weiyu; Wei, Linhua; Zhang, Shoutao; Feng, Lijie; Guo, Yanan

doi:10.1016/j.mimet.2022.106565

Cited by 3 publications

(4 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The parameters optimized for XE cocktail are summarized in Table 1. These results are consistent with data reported for SLiCE and ExoIII cloning by other groups (Dao et al, 2022;Gibson et al, 2009;Guo et al, 2022;Hsiao, 1993;Messerschmidt et al, 2016;Motohashi, 2015;Okegawa & Motohashi, 2015;Zhang et al, 2012), except for the use of ExoT and the dilution buffer. We finally developed a 2Â mix that assembles two DNA fragments with virtually any sequences in 5 min.…”

Section: Optimization Of Exo III Cloning Provides An Efficient Clonin...supporting

confidence: 92%

“…A subsequent study demonstrated that this extract may be prepared from any E. coli strains derived from K12, including mutants devoid of classical homologous recombination enzymes, such as RecA, RecB, and RecJ (Motohashi, 2015). Modifications and improvements to SLiCE preparation protocols have also been reported (Guo et al, 2022; Messerschmidt et al, 2016; Motohashi, 2015; Okegawa & Motohashi, 2015), leading to the establishment of systems for affordable DNA cloning. However, the molecular mechanisms underlying the assembly reaction in SLiCE remain unclear.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

et al. 2023

View full text Add to dashboard Cite

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double‐strand (ds) DNA‐dependent 3′‐5′ exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt‐ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3′ protruding ends; however, the addition of single‐strand DNA‐targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, “XE cocktail,” for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.

show abstract

Section: Optimization Of Exo III Cloning Provides An Efficient Clonin...supporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Data analysis tools, such as SAO, can extract critical technology information for layer mapping of data-driven TRM [95]. Initially, the SAO structure was widely used to analyze technical documents such as patents, present valid technical information, critical technocratic findings, and represent the relationships between technical elements [96][97][98][99][100][101][102]. Guo constructed SAO chains to identify future directions of technology [103].…”

Section: Subject-action-object Analysis With the Data-driven Technolo...mentioning

confidence: 99%

Data-Driven Technology Roadmaps to Identify Potential Technology Opportunities for Hyperuricemia Drugs

et al. 2022

Self Cite

View full text Add to dashboard Cite

Hyperuricemia is a metabolic disease with an increasing incidence in recent years. It is critical to identify potential technology opportunities for hyperuricemia drugs to assist drug innovation. A technology roadmap (TRM) can efficiently integrate data analysis tools to track recent technology trends and identify potential technology opportunities. Therefore, this paper proposes a systematic data-driven TRM approach to identify potential technology opportunities for hyperuricemia drugs. This data-driven TRM includes the following three aspects: layer mapping, content mapping and opportunity finding. First we deal with layer mapping.. The BERT model is used to map the collected literature, patents and commercial hyperuricemia drugs data into the technology layer and market layer in TRM. The SAO model is then used to analyze the semantics of technology and market layer for hyperuricemia drugs. We then deal with content mapping. The BTM model is used to identify the core SAO component topics of hyperuricemia in technology and market dimensions. Finally, we consider opportunity finding. The link prediction model is used to identify potential technological opportunities for hyperuricemia drugs. This data-driven TRM effectively identifies potential technology opportunities for hyperuricemia drugs and suggests pathways to realize these opportunities. The results indicate that resurrecting the pseudogene of human uric acid oxidase and reducing the toxicity of small molecule drugs will be potential opportunities for hyperuricemia drugs. Based on the identified potential opportunities, comparing the DNA sequences from different sources and discovering the critical amino acid site that affects enzyme activity will be helpful in realizing these opportunities. Therefore, this research provides an attractive option analysis technology opportunity for hyperuricemia drugs.

show abstract

“…[11][12][13][14][15] The second category encompasses methods such as ligation-independent cloning (LIC), Gibson assembly, and seamless ligation cloning extract (SLiCE). [16][17][18][19][20] While both categories are widely used in constructing DNA libraries, LIC or Gibson Assembly rely on multi-enzyme reactions, which can be relatively costly due to the need for multiple purified enzymes. In contrast, as previously stated, SLiCE utilizes the natural homologous recombination capabilities of E. coli cell lysates, offering a more economical alternative by using readily available laboratory strains.…”

Section: Introductionmentioning

confidence: 99%

RecombiCraft Library construction: A novel method for DNA Library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture

Kawai-Harada,

Mardikoraem,

Lauro

et al. 2024

Preprint

View full text Add to dashboard Cite

In this study, we introduceRecombiCraft, an innovative, rapid, and cost-efficient method for constructing DNA libraries inE. coli. This method uses seamless ligation cloning extract (SLiCE) coupled with liquid culture amplification to effectively minimize sequence biases. The technique capitalizes on the natural homologous recombination capabilities ofE. colicell lysates, eliminating the need for multiple purified enzymes and reducing costs. We first synthesized the library backbone and inserts via PCR, employing high-fidelity polymerase to minimize sequence bias. The SLiCE technique was then used to assemble the DNA fragments introduced intoE. colithrough electroporation. To ensure the integrity of the library, we optimized culture times based on next-generation sequencing analysis which confirmed the minimal sequence bias. The RecombiCraft method demonstrates that this approach is economical and maintains the library’s uniformity. It is a promising tool for genetic research and biotechnological applications with a significantly shorter library generation period.

show abstract

A variety of simple and ultra-low-cost methods preparing SLiCE extracts and their application to DNA cloning

Cited by 3 publications

References 13 publications

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

Data-Driven Technology Roadmaps to Identify Potential Technology Opportunities for Hyperuricemia Drugs

RecombiCraft Library construction: A novel method for DNA Library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture

Contact Info

Product

Resources

About