Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; Wehrs, Maren; Bailey, Constance B.; Kaplan, Nurgul; Hillson, Nathan J.; Mukhopadhyay, Aindrila; Keasling, Jay D.

doi:10.1038/s41598-018-20016-w

Cited by 40 publications

(39 citation statements)

References 31 publications

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“…Both ColE1 derivatives and RK2 origins have been shown to be targeted to specific subcellular locations near midcell (39). The pSC101 origin uses a Rep initiator protein to bind intron regions controlling replication and includes a partitioning locus to stabilize inheritance (40). In our previous study, we observed that plasmids with different origins were transferred by EVs at differing rates (12), although the plasmids compared previously were of various sizes.…”

mentioning

confidence: 93%

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Tran

Boedicker

2019

J Bacteriol

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Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles. IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.

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confidence: 93%

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Tran

Boedicker

2019

J Bacteriol

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“…In addition, we also optimized other terms, including the concentrations of L-arabinose and IPTG, the culture time, and the medium to further improve the system's performance (Table S6). At an appropriate concentration of L-arabinose, the expression levels of Cas9 and sgRNA were enough for cleaving the single-copy genome, but insufficient for cleaving all copies of plasmid#2 (about five copies [31]). Therefore, edited cells still possessed resistance to Amp.…”

Section: Optimization Of Crispr/cas9-assisted Recombineering Methodsmentioning

confidence: 99%

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

Huang

Guo

Wang

et al. 2020

Preprint

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Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

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“…Plasmid confirmation via MiSeq sequencing. Plasmid deep sequencing was performed as in previous work 50 . E. coli KCM competent cell prep.…”

Section: Methodsmentioning

confidence: 99%

Mevalonate Pathway Promiscuity Enables Noncanonical Terpene Production

et al. 2019

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Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into sixteen, seventeen and eighteen carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing sixteen carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13 C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel sixteen carbon terpenes by GC-QTOF. Based on the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

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Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

Cited by 40 publications

References 31 publications

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

Mevalonate Pathway Promiscuity Enables Noncanonical Terpene Production

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