EcoFlex: A Multifunctional MoClo Kit for <i>E. coli</i> Synthetic Biology

Moore, Simon; Lai, Hung-En; Kelwick, Richard; Chee, Soo Mei; Bell, David; Polizzi, Karen M.; Freemont, Paul S.

doi:10.1021/acssynbio.6b00031

Cited by 149 publications

(174 citation statements)

References 39 publications

Supporting

Mentioning

172

Contrasting

Order By: Relevance

“…Assembly of complete pathway consisting of all genes (RebF, RebH, RebO, RebD, VioA, VioB, VioC, VioD, VioE) was done using a modular cloning toolkit EcoFlex 32 , and is available on Addgene (Kit #1000000080). Briefly, all genes, including a kanamycin resistance marker KanR, were cloned into holding vector pBP-ORF, and then assembled into individual transcription units with standardised promoter (BBa_J23114 or BBa_J23108), pET-RBS and BBa_B0015 terminator.…”

Section: Cc-by-ncmentioning

confidence: 99%

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Lai¹,

Obled²,

Chee³

et al. 2017

Preprint

Self Cite

View full text Add to dashboard Cite

The next frontier in drug discovery could be the semi-synthesis of non-natural, xenobiotic compounds combining both natural product biosynthesis and synthetic chemistry. However, the required tools and underlying engineering principles are yet to be fully understood. One way to investigate non-natural product biosynthesis is to probe the substrate promiscuity of a clinically relevant biosynthesis pathway. Violacein is a bisindole compound produced by the VioABCDE biosynthesis pathway using L-tryptophan as the starting substrate. Previous studies have shown that violacein exhibits antimicrobial properties, and synthetic analogues of violacein might give rise to new targets for therapeutic development to combat antimicrobial resistance. By adding seven types of tryptophan analogues available commercially, 62 new violacein or deoxyviolacein analogues were generated with a synthetic violacein biosynthesis pathway expressed in Escherichia coli, demonstrating the promiscuity of violacein biosynthesis enzymes. Growth inhibition assays against Bacillus subtilis, a Gram-positive bacterium, were carried out to measure growth inhibitory activity of violacein analogues compared to violacein. In addition, we show that four new 7-chloro analogues of violacein or deoxyviolacein can be generated in vivo by combining the rebeccamycin and violacein biosynthesis pathways and purified 7-chloro violacein was found to have similar growth inhibitory activity compared to violacein. Structural studies of VioA revealed active site residues that are important for catalytic activity, and further pathway recombination with VioA homologues in related bisindole pathways may lead to more efficient enzymes that would accept tryptophan analogues more readily.

show abstract

Section: Cc-by-ncmentioning

confidence: 99%

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Lai¹,

Obled²,

Chee³

et al. 2017

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Golden Gate cloning is also frequently used for assembly of CRISPR gRNA expression arrays for site‐specific mutagenesis in a variety of organisms (McCarty, Shaw, Ellis, & Ledesma‐Amaro, 2019; Vad‐Nielsen, Lin, Bolund, Nielsen, & Luo, 2016). Finally, Golden Gate cloning has led to the development of the modular cloning system MoClo (Engler et al., 2014; Kowarschik, Hoehenwarter, Marillonnet, & Trujillo, 2018; Weber, Engler, et al., 2011; Werner et al., 2012) and of several other related modular cloning systems (Agmon et al., 2015; Andreou & Nakayama, 2018; Binder et al., 2014; Larroude et al., 2019; Lee, DeLoache, Cervantes, & Dueber, 2015; Martella, Matjusaitis, Auxillos, Pollard, & Cai, 2017; Moore et al., 2016; Occhialini et al., 2019; Pollak et al., 2019; Prielhofer et al., 2017; Sarrion‐Perdigones et al., 2011, 2013).…”

Section: Commentarymentioning

confidence: 99%

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Marillonnet

Grützner

2020

CP Molecular Biology

View full text Add to dashboard Cite

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level –1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors

show abstract

“…The core of GG strategy lies in establishing a library of standardized and interchangeable DNA parts, which can be subsequently assembled in a single-step, one-pot reaction. Examples of such GG platforms have been recently reported for Escherichia coli, yeast or plant species (Engler et al, 2014;Terfr€ uchte et al, 2014;Agmon et al, 2015;Kakui et al, 2015;Lee et al, 2015;Mitchell et al, 2015;Iverson et al, 2016;Moore et al, 2016). Nevertheless, many of valuable biotech workhorses are still queuing the line for a customized GG platform, amongst these the non-conventional yeast Y. lipolytica, which is a wellestablished biotechnological chassis for the production of numerous valuable bioproducts (Nicaud, 2012;Ledesma-Amaro et al, 2015;Madzak, 2015;Ledesma-Amaro and Nicaud, 2016).…”

Section: Introductionmentioning

confidence: 99%

Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica

Celińska

Ledesma-Amaro

Larroude

et al. 2017

Microbial Biotechnology

111

118

View full text Add to dashboard Cite

SummaryIn this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements. Previously validated DNA modules (regulatory elements and selection markers) were adopted as the Golden Gate bricks. The system's operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.

show abstract

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

Cited by 149 publications

References 39 publications

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica

Contact Info

Product

Resources

About