Plasmid purification by using a new naphthalene tripodal support

Streptococcus agalactiae(group BStreptococcus[GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shownin vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried theaacA-aphDgene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3family transposon. Its ability to confer HLGR was proven by transfer into anEnterococcus faecalisisolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformedE. faecalisstrain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally.

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SpinSmart Plasmid Purification Protocols v1

protocols.io ◽

10.17504/protocols.io.grjbv4n ◽

2016 ◽

Author(s):

Denville Scientific

Keyword(s):

Plasmid Purification

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SpinSmart Plasmid Purification Protocol: High-copy plasmid DNA from E. coli v1

protocols.io ◽

10.17504/protocols.io.gqibvue ◽

2016 ◽

Author(s):

Denville Scientific

Keyword(s):

Plasmid Dna ◽

E Coli ◽

Plasmid Purification ◽

Purification Protocol ◽

High Copy Plasmid

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Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Microbial Cell Factories ◽

10.1186/s12934-021-01642-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ram Shankar ◽

Nina Schäffer ◽

Marco Schmeer ◽

Joe Max Risse ◽

Karl Friehs ◽

...

Keyword(s):

Escherichia Coli ◽

Cation Exchange ◽

Plasmid Dna ◽

Recombinant Expression ◽

Exchange Chromatography ◽

Dna Purification ◽

Specific Productivity ◽

Scale Production ◽

Cation Exchange Chromatography ◽

Plasmid Purification

Abstract Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

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Automated Filtration-Based High-Throughput Plasmid Preparation System

Genome Research ◽

10.1101/gr.9.5.463 ◽

1999 ◽

Vol 9 (5) ◽

pp. 463-470 ◽

Cited By ~ 4

Author(s):

Masayoshi Itoh ◽

Tokuji Kitsunai ◽

Junichi Akiyama ◽

Kazuhiro Shibata ◽

Masaki Izawa ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Microtiter Plate ◽

Linear Process ◽

Alkaline Lysis ◽

Filtration Method ◽

Cell Harvesting ◽

Automated Processing ◽

Plasmid Preparation ◽

Plasmid Purification

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

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