1. Glycine in growth medium
Evaluation of Electroporation Efficiency of Lactobacillus sobrius, Lactobacillus Plantarum, Lactobacillus sakei subsp. sakei 2a and Lactococcus lactis MK02
KRUGER, Monika F.1,3; BRON, Peter2; PEREZ, Odette3; SAAD, Susana I.4; FRANCO, Bernadette D.G.M.1; SMIDT, Hauke3; ZOETENDAL, Erwin3
1 Universidade de São Paulo, Food Microbiology - São Paulo, Brazil; 2 NIZO Food Research, – Ede, The Netherlands; 3 Wageningen University and Research Centre, Molecular Ecology - Wageningen, NL; 4 Universidade de São Paulo, Food Technology - São Paulo, Brazil.
Results
The treatment with 2% of glycine extended the Lag phase of growth in 1.5-h, approximately for all tested strains, and L. sakei 2a and L. plantarum are not able to growth at 3% of glycine. The optimum transformation efficiency was achieved for L. plantarum using 0% of glycine following the protocol A (7.4 x 104 cfu/µg), as showed in Table 1.
Table 1: Electrotransformation efficiency of LAB strains with pNZ123 at different glycine concentration using two different protocols (A and B). The transformants on MRS agar plates containing 20 µg ml-1 Cm were determined after a 48-h incubation at 37°C
Introduction
There is a considerable interest in the manipulation and improvement of lactic acid bacteria due to the widespread biotechnological application of these genera in food and medical industry [1]. Electroporation protocols and media can have a major impact on post-electroporation cell viability and transfection efficiency [1]. In this study we evaluated the generation of competent cells and the transformation efficiency of four lactic acid bacteria strains (LAB): L. sobrius and L. plantarum isolated from piglet Intestine [3] in Laboratory of Microbiology - WUR, NL.; L. sakei 2a isolated from fresh pork sausage [4] and L. lactis MK02 [5] isolated from fresh rocket salad in the Laboratory of Food Microbiology - USP,BR.
Materials and Methods
Electrocompetent cells (Figure 1) were prepared following the protocol A [1], and protocol B [2]. The transformation efficiency was defined as the number of viable cells of transformants per microgram of plasmid DNA.
PCR amplification of Cmr gene was made to detect and validate the presence of pNZ 123 plasmid in the transformants cells (Figure 2), according to Kim et al., 2005.
Strain
Protocol
Glycine in growth medium
0 %
1 %
2 %
3 %
Transformants /µg of pNZ123
L. sobrius
(A) PEG 30%
4.43 x 103
7.62 x 103 -
(B) MgCl2
L. plantarum
1.9 x 103
0.6 x 101
1.79 x 102 ng
7.4 x 104
L. lactis
5.3 x 102
2.38 x 102
L. sakei 2a
(a) PEG 30%
(b) MgCl2
ng: no growth in culture media
Figure 2: 1% Agarose gel electrophoresis of PCR products. 1-kbp DNA ladder marker (Gibco-BRL, lane 1), pNZ123 (lane 2), wild-type LAB (lanes 3 and 4) and transformants L. sobrius, L. plantarum, L. sakei 2a and L. lactis MK02 (lanes 5,6,7 and 8 respectively)
MRS broth
Culture
37° C – 18hs
Pre-culture
37 °C – 18hs
Electroporation
Post-Electroporation Recovery
37° C – 2 to 3hs
Plasmid: pNZ 123 (4µL)
Pulse strength: 1.5 kV cm-1, 25 µF, 400 Ω
Time constant: 9.0 – 9.6
MRS broth M sucrose + 0.1M MgCl
MRS + 1% glycine
MRS + 2% glycine
Recovery
37° C - 48hs
MRS Agar + 0.5M sucrose + 0.1M MgCl + 20µL mg-1
MRS + 0% glycine
MRS + 3% glycine
Reserved for PCR picture
Conclusion: High transformation efficiency was very dependent on the concentration of glycine in the growth media. Another transformation protocol should be developed to the optimization of the transformation efficiency of the strains tested in this study.
Figure 1: Culture, Electroporation and Recovery Procedure
References:
[1] Kim et al., (2005) J Appl Microbiol 99: 167–174;
[2] Goffin et al., (2005) J Bacteriol 187:6750–6761;
[3] Konstantinov et al., (2006) Int J Syst Evol Microbiol 56: 29-32;
[4] De Martinis & Franco, (1998) Int J Food Microbiol 42: 119–126;
[5] Kruger et al., (2009) in preparation
Contact
Wageningen UR: Phone: NL (31)
Universidade de São Paulo: Phone: BR (55)
Kruger, M.F.: Phone: NL (31) ; BR (55)