Novel plasmid transformation method mediated by chrysotile, sliding friction, and elastic body exposure

Our plain-English summary of Novel plasmid transformation method mediated by chrysotile, sliding friction, and elastic body exposure, by N. Yoshida, T. Nakajima-Kambe, K. Matsuki, et al (Analytical Chemistry Insights, 2007).

The study

• Authors: N. Yoshida, T. Nakajima-Kambe, K. Matsuki, T. Shigeno
• Journal: Analytical Chemistry Insights
• Published: 2007;2:9-15
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Free full text

Abstract

Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 10(7) per microg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.

In plain English

This laboratory experiment described and optimised a bacterial transformation method in which Escherichia coli cells were suspended in a chrysotile colloidal solution containing plasmid DNA (10-50 ng of pUC18), spread on the surface of an elastic body (2% LB agar), and rubbed for 60 seconds with a polystyrene streak bar on a turning table, a procedure the authors called elastic body exposure. The chrysotile fibers used were 95% of 50-70 nm diameter and 0.34-2.30 micrometres length. Transformants were counted as ampicillin-resistant colonies. The authors reported that transformation efficiency was enhanced with a cell density of optical density 2 or more at 550 nm, with a vertical reaction force of 40 g or more (double that at 20 g), and with rotation speeds exceeding 30 rpm, reaching 10 to the seventh transformants per microgram of plasmid DNA. Cells grown on LB plates gave 5-6 x 10 to the sixth transfectants per microgram versus 10 to the sixth for broth-grown cells. The authors concluded the method is simple, reliable and reproducible, and that transformation and plating are performed simultaneously.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experiment in bacteria (methods-development and optimisation study)
• Who was studied: Escherichia coli strains JM109 (mainly), DH5-alpha and C600, cultured on LB agar plates or in LB broth at 37 degrees C; no human or animal subjects
• Exposure: Chrysotile whisker colloidal solution (fibers 95% with diameters 50-70 nm and lengths 0.34-2.30 micrometres) mixed with plasmid DNA and bacteria, then subjected to sliding friction on agar
• Main results:
  • Recipient cell density of optical density 2 or more at 550 nm in the chrysotile-plasmid cell mixture increased transformation efficiency; few ampicillin-resistant colonies were obtained at optical density 1 or less
  • A vertical reaction force of 40 g or more on the streak bar doubled transformation efficiency compared with 20 g
  • A marked reduction in transformation efficiency occurred when rotation speed was reduced to 10 rpm; high efficiency was seen at rotation speeds exceeding 30 rpm
  • Under the optimal conditions the authors observed a transformation efficiency of 10 to the seventh per microgram of plasmid DNA
  • E. coli JM109 grown in LB broth gave 10 to the sixth transfectants per microgram of pUC18, versus 5-6 x 10 to the sixth for cells grown on LB plates
  • Other E. coli strains: DH5-alpha 1.3 x 10 to the sixth and C600 1.8 x 10 to the sixth transformants per microgram of pUC18 DNA
  • Friction coefficient at the bar-agar interface rose sharply once the surface dried after eight or nine turns (vertical force fixed at 40 g)
• The authors' conclusion: The authors concluded that their elastic body exposure method is simple, reliable and highly reproducible, and that rapid drying of the plate surface is essential for transformation by this method
• Limitations the authors state:
  • The authors noted an upper limit on the applied force, since more than 100 g of vertical reaction force was likely to destroy the elastic body

Topics

Medical terms in this study, explained in our Lexicon: Chrysotile.

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