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How to make comp. P. Aeruginosa

Best

Benchtop and Microcen-

trifuge Preparation of

Pseudomonas aeruginosa

Competent Cells

BioTechniques 33:760-763 (October 2002)

Although indispensable for the intro-

duction of DNA fragments manipulated

in vitro into bacteria, transformation us-

ing chemically prepared competent or

electrocompetent cells is still a rather

slow and labor-intensive technique, and

apart from waiting for transformants to

appear on selective plates, the prepara-

tion of competent cells is the slowest

part of the whole process. Most pub-

lished transformation protocols for use

with Pseudomonas aeruginosa are mod-

ifications of protocols developed for E.

coli and either involve electrocompetent

cells (1–4,9,12) or cells made competent

by chemical treatment (8,10). Conven-

tional chemical transformation of P.

aeruginosa has many drawbacks, most

notably low transformation efficiencies

and/or time-consuming procedures.

Therefore, electroporation has become

the method of choice for this bacterium.

However, the preparation of low-ionic

strength DNA and competent cells for

electroporation is still a critical and rel-

atively time-consuming process. Here

we describe the adaptation of a rapid

benchtop- and microcentrifuge-based

method previously reported (14) for ob-

taining low-efficiency competent E. coli

cells to prepare highly competent P.

aeruginosa cells for chemical transfor-

mation. For P. aeruginosa, this method

yields transformation efficiencies that

are comparable to those observed when

using electroporation.

For preparation of competent P.

aeruginosa cells, strain PAO1 cells

were grown in 4 mL LB broth (Difco

Laboratories, Detroit, MI, USA) at

37°C until they reached saturation

(usually overnight). All of the follow-

ing steps, except the centrifugations,

were performed with the tubes sitting

on ice. A 1.5-mL microcentrifuge tube

was pre-chilled on ice for 3–5 min.

Aliquots (1 mL) of a stationary phase

or overnight P. aeruginosa culture were

transferred to the chilled microcen-

trifuge tubes, and the cells were har-

vested at room temperature by a short

centrifugation for 30 s at approximate-

ly 13 000× g. Centrifugations longer

than 30 s are not recommended because

they cause significant reduction of

transformation efficiencies. The cell

pellets were resuspended with a pipet

tip in 1 mL cold (approximately 4°C)

0.1 M MgCl2. The cell suspension was

again centrifuged at approximately

13 000× g for 30 s at room temperature.

The supernatant was removed, and the

cell pellets were resuspended with a

pipet tip in 1 mL cold transformation

salts with glycerol (TG salts) solution

(75 mM CaCl2, 6 mM MgCl2, 15%

glycerol). The cell suspensions were

kept on ice for 10 min and then cen-

trifuged at approximately 13 000× g for

30 s at room temperature. After decant-

ing the supernatant, the pellets were

then resuspended with a pipet tip in 200

µL cold TG salt solution and kept on

ice until use. At this stage, the cells

were ready for transformation.

The transformation efficiency of P.

aeruginosa cells prepared using this

procedure was determined using the

4.2-kb broad-host-range plasmid pUC-

P20T (11). Aliquots (100 µL) of the

competent cells were transferred to

thin-walled 13 × 100 mm borosilicate

glass tubes (VWR Scientific, South

Plainfield, NJ, USA) that were pre-

chilled on ice. Next, 2–5 µL aliquots

containing 100–1000 ng pUCP20T

DNA that has been purified using the

Qiagen® Midiprep kit (Qiagen, Valen-

cia, CA, USA) and suspended in either

10 mM Tris-HCl (pH 8.5) or sterile wa-

ter were added to the cells (DNA in this

buffer or water yielded the same trans-

formation efficiencies). A negative con-

trol received sterile water instead of

plasmid DNA. This DNA-cell mixture

was incubated on ice for 15 min. The

mixture was then heat-shocked at 37°C

for 2 min, and 500 µL LB broth were

immediately added. The tubes were in-

cubated at 37°C for 1 h in a shaking in-

cubator. After incubation, a 200-µL

aliquot of each cell suspension was

plated on LB agar containing 200

µg/mL carbenicillin (Gemini Bio-Prod-

ucts, Woodland, CA, USA). The re-

maining contents of each tube were

transferred to microcentrifuge tubes,

and cells were harvested by centrifuga-

tion at approximately 13 000× g for 1

min at room temperature, resuspended

in 200 µL LB medium, and then spread

on a LB-carbenicillin plate. The plates

were incubated at 37°C for 20–24 h be-

fore colonies were counted.

Using the procedures described

above for the preparation and transfor-

mation of competent P. aeruginosa

PAO1 cells (Figure 1), we obtained 1 ×

105 transformants/µg pUCP20T DNA

(Table 1), using an optimal DNA con-

centration of 200–400 ng. This trans-

formation efficiency is comparable to

the 1.1–1.5 × 105 transformants/µg

DNA when using most electroporation

protocols (1–4,9,12). Only two other

reports indicated higher transformation

efficiencies [i.e., 1.2 × 106 transfor-

mants/µg DNA by using chemically

prepared competent cells and a heat

shock at 50°C (8) and approximately 2

× 106 transformants/µg DNA by em-

ploying an electroporation procedure

(3)]. Although the transformation effi-

ciency of competent cells prepared us-

ing our procedure is about 10-fold low-

er when compared to the most efficient

procedures, it is comparable to those

achieved with most published methods

and therefore more than adequate for

most laboratory applications. However,

Benchmarks

Vol. 33, No. 4 (2002)

You can also use the calcium chloride technique. It is simple and straight method.

You can also use the calcium chloride technique. It is simple and straight method.

Thank you very much