Rambling and Scrambling in Bacterial Transformation— a Historical and Personal Memoir

Lacks, Sanford A.

doi:10.1128/jb.185.1.1-6.2003

Cited by 9 publications

(6 citation statements)

References 55 publications

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“…From early groundbreaking experiments on bacterial transformation 3 , to the most recent advances in genomic engineering using targetable nucleases 4,5 , a wide array of tools have been developed in order to introduce and maintain exogenous genetic material in an organism or to directly edit the existing genome. However, while multiple strategies are often available for the majority of model organisms, many species of biological interest still lack efficient genetic engineering tools 6 .…”

Section: Introductionmentioning

confidence: 99%

CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system

Ruiz

Talenton

Dubrana

et al. 2019

Preprint

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Over the last decade a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or “cloning”) their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria and algae. The cloning step requires the insertion of yeast genetic elements within the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-Cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast’s highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

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Section: Introductionmentioning

confidence: 99%

CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system

Ruiz

Talenton

Dubrana

et al. 2019

Preprint

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“…Besides the formation of a supramolecular scaffold, the asymmetric insertion of a membrane protein into one leaflet of the lipidic bilayer can lead to a modification of the membrane curvature. Such a mechanism has been hypothesized for monotopic proteins inducing membrane proliferation such as MurG [ 137 ], LpxB [ 138 ] and PtmA [ 139 ] and confirmed by sequence comparison and in silico studies of alMGS [ 140 , 141 ]. In addition, PtmA and alMGS have been purified and their ability to remodel synthetic liposomes into lipid tubules has also been demonstrated in vitro [ 139 , 141 , 142 ].…”

Section: Mechanisms Of Protein-induced Membrane Curvaturementioning

confidence: 72%

“…Finally, alMGS overproduction also activates the σ E and Cpx envelope stress responses, which can activate phospholipid metabolism as discussed in “ The importance of phospholipid homeostasis ” section. Besides alMGS, all of the monotopic membrane proteins listed in Table 1 (MurG, LpxB, caveolin-1 and PmtA) are known to bind to the lipid bilayer via electrostatic interactions with anionic phospholipids [ 104 , 137 – 139 ]. Electrostatic interactions with transmembrane proteins have been less studied.…”

Section: Influence Of Membrane Curvature On Phospholipid Biosynthesismentioning

confidence: 99%

“…Possible oligomerization? – [ 97 ] Caveolin-1 M H. sapiens E. coli Vesicles Well-defined supramolecular cage (160 monomers per cage) PG and lysophospholipids enrichment (not quantified) [ 104 , 105 ] MurG M E. coli E. coli Vesicles – CL enrichment (~ 22% mol) [ 137 ] LpxB M E. coli and H. influenzae E. coli Tubules and vesicles – PG and CL enrichment (not quantified) [ 138 ] PmtA M A. tumefacensis A. tumefacensis Vesicles – CL needed for membrane proliferation (not quantified) [ 139 ] alMGS M A. laidlewii E. coli Vesicles – PG and CL enrichment (not quantified) [ 140 – 142 , 193 ] …”

Section: Introductionmentioning

confidence: 99%

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Inducible intracellular membranes: molecular aspects and emerging applications

et al. 2020

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Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering an abnormal accumulation of membrane structures inside the cells (or membrane proliferation) share two major common features: (1) they promote the formation of highly curved membrane domains and (2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, and inspire new applications, either for academia (high-yield membrane protein production and nanovesicle production) or industry (biofuel production and vaccine preparation).

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“…Sandy has been a leader in defining genetic transformation in Streptococcus pneumoniae for the last 40 or more years. His talk at the conference is presented elsewhere in this issue (4). Also, the continual good advice of Phil Harriman (National Science Foundation) was noted and recognized.…”

mentioning

confidence: 99%