We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

Rapid and simple method for purification of nucleic acids.

MUTATION PREVENTING FORMATION OF HEPATITIS B e ANTIGEN IN PATIENTS WITH CHRONIC HEPATITIS B INFECTION

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

Delivery of CRISPR-Cas nucleases using bacteriophage enables targeted killing of microbes in a population

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

The bacterial AlkB protein is known to be involved in cellular recovery from alkylation damage; however, the function of this protein remains unknown. AlkB homologues have been identified in several organisms, including humans, and a recent sequence alignment study has suggested that these proteins may belong to a superfamily of 2-oxoglutarate-dependent and iron-dependent oxygenases (2OG-Fe(ii)-oxygenases)1. Here we show that AlkB from Escherichia coli is indeed a 2-oxoglutarate-dependent and iron-dependent DNA repair enzyme that releases replication blocks in alkylated DNA by a mechanism involving oxidative demethylation of 1-methyladenine residues. This mechanism represents a new pathway for DNA repair and the third type of DNA damage reversal mechanism so far discovered.

AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

https://www.pnas.org/content/pnas/86/7/2172.full.pdf

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

Several effective methods for bacterial cell transformation have previously been described. These methods involve treatment of cells with CaCl2 (1-5) or cobalt hexamine chloride (6). Agents such as polyethylene glycol (PEG) have also been shown to induce bacterial cell transformation (7). We now describe a new method for the preparation of competent bacterial cells that yields high transformation efficiencies but is simpler and more convenient than other techniques. Bacterial cells are grown to the early log phase (ODgrjo = 0.3-0.6) in LB broth, then pelleted by centrifugation (1,000 X g for 10 mins at 4°C). The cells are then resuspended in l/10th volume of transformation and storage buffer (TSB): LB broth (pH 6.1) containing 10% PEG (MW-3,350), 5« DMSO and 20 mH Mg + + (lOmM MgCl2 + 10 mH MgSO4) at 4°C, and incubated on ice for approximately 10 mins. For transformation, 0.1 ml aliquots of the cells are pipetted into cold polypropylene tubes and mixed with 100 pg of plasmid DNA. The cells are returned to ice for 5-30 mins. Heat shock is not necessary. Next, cells are grown to permit expression of the antibiotic resistance gene [0.9 ml of TSB with 20 mM glucose is added, and the cells incubated at 37°C with shaking (225 rpm) for 60 mins] and plated on antibiotic-containing agar plates for selection of transformants. Using this protocol, we obtain transformation efficiencies of up to 2 X 10 transformants per microgram of DNA. Moreover, cells in TSB can be frozen (in a dry ice/ethanol bath) and stored at -70°C for use at a later date without a significant loss of cell viability or transformation efficiency. This technique provides a convenient and reproducible method for the preparation and storage of competent bacterial cells.

A rapid and convenient method for the preparation and storage of competent bacterial cells

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.1840090226

Compact organization of the hepatitis B virus genome.

Publisher Summary The uptake of foreign DNA by Escherichia coli can be induced either through electroporation, which involves discharging an electrical voltage across bacterial cell membranes, or by making bacteria competent through chemical methods. Two widely used chemical methods involve treating bacteria with calcium chloride or hexamine cobalt and subjecting the cells to a heat shock. The protocol developed for the preparation of competent E. coli was designed to be rapid, convenient, and inexpensive, yet suitable to achieve transformation efficiencies sufficient for routine cloning experiments. Although transformation efficiencies obtained with the protocol are comparable to those of other commonly used methods to prepare competent bacteria, the technique is advantageous in that it is simple and can be completed in a short time once the bacteria are grown. Although the conditions necessary to achieve maximum numbers of transformants are specified, there is flexibility in the assay.

Preparation and storage of competent Escherichia coli cells.

Wehavedeveloped asimple, one-step proce- dureforthepreparation ofcompetentEscherichia coli that uses atransformation andstorage solution (TSS; lxTSSisLB brothcontaining i0%(wt/vol) polyethylene glycol, 51% (vol/vol) dimethyl sulfoxide, and50mM Mg2+atpH6.5). Cells aremixedwithanequal volume ofice-cold 2xTSSand areimmediately readyforuse.Genetic transformation is equally simple: plasmid DNA isaddedandthecells are incubated for5-60minat40C.A heatpulse isnotnecessary andtheincubation timeat40Cisnotcrucial, sothere areno critical timing steps inthetransformation procedure. Trans- formed bacteria aregrownandselected bystandard methods. Thus,this procedure eliminates thecentrifugation, washing, andlong-term incubation steps ofcurrent methods. Although cells taken early inthegrowth cycle (OD6m 0.3-0.4) yield the highest transformation efficiencies (107-108 transformants per jug ofplasmid DNA),cells harvested atother stages inthe growthcycle (including stationary phase) arecapable of undergoing transformation (105-107 transformants perjpg of DNA).Forlong-term storage ofcompetent cells, bacteria can befrozen inTSSwithout addition ofother components. Our procedure represents asimple andconvenient method forthe preparation, transformation, andstorage ofcompetent bacte- rial cells. Several chemical methods havebeenestablished that induce bacterial cell transformation. Inaclassic experiment, Mandel andHiga(1) demonstrated that treatment ofEscherichia coli withCaCl2 madethecells susceptible touptake ofbacterio- phage DNA.Subsequently, itwasshownthat this technique could beusedtotransform E.coli withbacterial chromo-

/pdf/one-step-preparation-ofcompetent-escherichia-coli-1ohopb5zet.pdf

C. T. Chung

Papers

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

A rapid and convenient method for the preparation and storage of competent bacterial cells

Compact organization of the hepatitis B virus genome.

Preparation and storage of competent Escherichia coli cells.

One-step preparation ofcompetent Escherichia coli: Transformation andstorage ofbacterial cells inthesamesolution