High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol

Papagianni, Maria; Avramidis, Nicholaos; Filioussis, George

doi:10.1186/1472-6750-7-15

Cited by 45 publications

(26 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…LiAc was proposed to increase the permeability of yeast cell wall. Recently, LiAc was reported to increase the electroporation efficiency in bacterium (Papagianni et al, 2007). Here, we tested whether LiAc can be used in CHO cell transient transfection to improve transfection efficiency.…”

Section: Liac Increased Protein Expression Levelsmentioning

confidence: 98%

See 1 more Smart Citation

High‐level protein expression in scalable CHO transient transfection

Kober

Tellers

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14-day fed-batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed.

show abstract

Section: Liac Increased Protein Expression Levelsmentioning

confidence: 98%

“…Therefore, 3 mM LiAc addition was chosen as the optimal condition. Notably, the LiAc concentration used in yeast and bacteria was much higher than that used in CHO cells (100 mM in yeast versus less than 10 mM in CHO cells) (Gietz et al, 1995;Papagianni et al, 2007).…”

Section: Liac Increased Protein Expression Levelsmentioning

confidence: 99%

High‐level protein expression in scalable CHO transient transfection

Kober

Tellers

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Plasmids from positive candidates were sequenced (Sourcebioscience UK) using primers pORI280FOR (5=CTCGTTCATTATAACCCTC 3=) and pORI280REV (5=CGCTTCCTTTCCCCCCAT3=) to verify the deliberate mutation and to confirm that no other changes had been introduced. pDF08 (pORI280-nisM21V) was then introduced into L. lactis DGCC 10042 pVE6007 by electroporation (22), and transformants were selected by growth on GM17-Ery-X-Gal plates at 30°C. Integration of pDF08 by single-crossover recombination and curing of the temperaturesensitive plasmid pVe6007 were achieved by growth at 37°C in GM17-Ery broth and plating on GM17-Ery-X-Gal agar at the same temperature.…”

Section: Methodsmentioning

confidence: 99%

Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

Field

Daly

O’Connor

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 g/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes. The growing consumer demand for food products that are minimally processed and free of chemical preservatives presents a difficult challenge for food processors. Consequently, there has been a focus on the application of naturally produced antimicrobial compounds as a more acceptable means to control the growth of undesirable microorganisms in food (1, 2). Bacteriocins (ribosomally produced, small, heat-stable peptides that are active against other bacteria) derived from organisms generally regarded as safe provide one potential solution. However, only two bacteriocins have been made commercially available to any extent. These are nisin, produced by Lactococcus lactis, and pediocin PA-1, produced by Pediococcus acidilactici (3, 4). Of these, nisin is used in a wide variety of dairy and nondairy products, including cream and cheese products, soups, liquid egg, mayonnaises, salad dressings, tomato products, and beer (5). Nisin A exhibits antibacterial activity against a wide range of Gram-positive bacteria, including food-borne pathogens such as staphylococci, bacilli, clostridia, and Listeria (6, 7). Indeed, the success of nisin A from discovery (8) through to regulatory approval and finally to commercial application has spurred researchers to exploit its geneencoded nature and to attempt to "bioengineer" variants with altered biological, chemical, and physical properties. Over the last decade, several studies have described the discovery of new nisi...

show abstract

“…Although low salts buffer is usually used to remove these salts, the use of a nonionic buffer such as glycerol would be more preferable. Buffers such as water (Enderle and Farwell, 1998;Papagianni et al 2007), water and glycerol (Sheng et al 1995;Sharma and Schimke, 1996), or salts buffer and glycerol have also been widely used (Dower et al 1988). However, glycerol is seldom used alone as a buffer (Dorella et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Enhancing DNA electrotransformation efficiency in Escherichia coli DH10B electrocompetent cells

Wu¹,

Matand²,

Kebede³

et al. 2010

Electron. J. Biotechnol.

View full text Add to dashboard Cite

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10 9 cfu/µg by washing cells three times with 15 ml of 10% glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10 10 and 0.6 x 10 10 cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.

show abstract

High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol

Cited by 45 publications

References 17 publications

High‐level protein expression in scalable CHO transient transfection

High‐level protein expression in scalable CHO transient transfection

Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

Enhancing DNA electrotransformation efficiency in Escherichia coli DH10B electrocompetent cells

Contact Info

Product

Resources

About