One-Step Cloning and Chromosomal Integration of DNA

St-Pierre, François; Cui, Lun; Priest, David G.; Endy, Drew; Dodd, Ian B.; Shearwin, Keith E.

doi:10.1021/sb400021j

Cited by 192 publications

(198 citation statements)

References 19 publications

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“…The pFC25 vector was constructed by use of Gibson assembly using a hygromycin-resistant version of the replicable vector pCM158 as a backbone (20). The hygromycin resistance marker (Hm r ) was amplified by PCR from plasmid pTEC27 (Addgene accession number 30182) (21), the flippase gene was amplified by PCR from plasmid pE-FLP (Addgene accession number 45978) (22), and P tac was amplified from pAWP89 (9). To create the insertion PCR product containing the pmo promoter fused to xylE, two PCR products were generated for the flanking integration sites and correspond to the intergenic region and coding sequences of the genes METBUDRAFT_2794 and METBUDRAFT_2795.…”

Section: Methodsmentioning

confidence: 99%

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Yan

Chu

Puri

et al. 2016

Appl Environ Microbiol

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Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains. Methane, the principal component of natural gas and biogas, is a cheap, abundant, and renewable energy and carbon source. However, methane is also the second most prevalent greenhouse gas (1). Therefore, technologies to efficiently convert methane to chemicals or fuels can bring new sustainable solutions to a number of industries with large environmental footprints. Methane-oxidizing bacteria (methanotrophs) are able to use methane as their sole source of carbon and energy and thus are promising systems for methane-based bioconversion (2, 3). The bioconversion of methane to industrial products (single-cell proteins, biopolymers, lipids, etc.) using aerobic methanotrophs has been studied for approximately 50 years but has had little enduring success (4). Current biological engineering and systems biology approaches provide new opportunities for metabolic engineering of methanotrophs. However, to be an industrial workhorse like Escherichia coli, an industrial methanotroph needs to have a high growth rate, and efficient genetic tools for its manipulation and a fundamental knowledge base are needed.Many methanotrophs have been isolated and characterized since the classic study of Whittenbury et al. (5). Aerobic methanotrophs are found in two major groups, the...

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Section: Methodsmentioning

confidence: 99%

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Yan

Chu

Puri

et al. 2016

Appl Environ Microbiol

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“…The lacZ reporters and LacI and CI expression constructs were chromosomally integrated using the OSIP system and its precursors (23,39) into MG1655 rph + ΔlacIZYA (SI Materials and Methods). Cells were grown at 37°C in minimal medium (LacI looping strains) or in rich medium (CI looping strains) and assayed by a modified LacZ microtiter plate method (7) (SI Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Priest

Cui

Kumar

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to longrange DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250-to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5-to 10-kb distances, was able to increase contact probability 10-to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5-to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells. j factor | TPM | promoter-enhancer I nteractions between proteins bound to separate sites on the same DNA molecule are critical in gene regulation and other DNA processes (1-4). The DNA separation between functionally interacting sites ranges from a few base pairs to hundreds of kilobase pairs, as in the case of some eukaryotic enhancers and their promoters. At short separations, it is clear that the DNA acts as a tether that keeps one site in the vicinity of the other so that the proteins at one site can find the other site in 3D space more efficiently than if they were free in solution (Fig. 1). Tethering is also a way to provide specificity because it aids interaction with linked sites but not unlinked sites. However, as the separation between the sites increases, this tethering effect becomes weaker, and it is not understood how the DNA linkage between widely separated sites, for example, between enhancers and promoters, provides the efficiency and specificity required for proper regulation.The effect of DNA tethering can be quantified by the factor j LOOP (M), the effective molar concentration of one site on the DNA relative to the other, or as the free energy of the DNA looping reaction ΔG LOOP (Fig. 1) (5-9). These parameters are interconvertible: ΔG LOOP = -RTlnj LOOP (kcal/mol; the reference j is 1 M). The formation of a naked DNA loop is in itself an energetically unfavorable reaction (ΔG LOOP is positive) under physiological conditions due to the enthalpic cost of DNA bending and twisting (particularly important for s...

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“…The kit, however, failed for the ALE-3 strains, and the DE3 cassette was therefore amplified from BL21-DE3 strain using USER primers (T7LacI_UF and UR) cloned in pOSIP-KO (St-Pierre et al, 2013) by amplifying the plasmid with pOSIP_UF and UR (Table S2, Supplementary information) for genome integration followed by loop out of integration cassette with pE-FLP. The plasmids for overexpression of the serine pathway (pCDFDuet-1-serAmut-serC and pACYCDuet-1-serB) (Mundhada et al, 2016) were transformed in the ALE-3-2(DE3), ALE-4-2(DE3) and ALE-5(DE3) strains respectively.…”

Section: De3 Integration and Batch Fermentations Of Ale Strains For Smentioning

confidence: 99%

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

Mundhada

Seoane

Schneider

et al. 2017

Metabolic Engineering

121

141

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L-serine is a promising building block biochemical with a high theoretical production yield from glucose.Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Other mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium.Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology. AbbreviationsAdaptive Laboratory Evolution (ALE); Mutiplex Genome Engineering (MAGE).

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One-Step Cloning and Chromosomal Integration of DNA

Cited by 192 publications

References 19 publications

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

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