Archive \ Volume.11 2020 Issue 3

 

An improved method for induction of competency in Escherichia coli DH5α

 

Vajiheh Eskandari

 

Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran.

 


Abstract

Efficient DNA transformation of bacterial competent cells has a critical role in biotechnology experiments such as gene cloning and protein expression. Transformation rates are commonly associated with the competency state of the bacterial cells. In this paper, it was demonstrated that transformation efficiency is strongly related to the growth phase of bacterial culture used for competent cell preparation, as well as special chemical treatments applied to make them competent. Results showed that exposure of the cells from the late growth phase (OD600; 0.9-0.94) to monovalent and divalent cations resulted in the highest transformation efficiencies (about 100-fold). These competent cells are particularly useful for the transformation of ligated and large DNA molecules (more than 10 kbp).

Keywords: Monovalent cations, Competent cells, Transformation, E. coli


INTRODUCTION

 

Plasmid transformation into bacterial competent cells is a critical stage of gene manipulation in molecular cloning. In the early 1970s, Cohen [1] showed that recombinant plasmids can be assimilated by E. coli cells using a calcium chloride treatment. So far, several chemical methods have been developed for increasing the competency of the E. coli. A number of factors such as bacterial strain, medium composition, growth phase, both monovalent and divalent cations, and DNA size are important in the chemical transformation process [2]. Actively growing cells in early and mid-exponential phases are more susceptible to transformation [3]. Previous studies have reported that the cultures grown to mid-exponential phase (OD600 nm at 0.4) seem to give a high transformation rate for E. coli indicating the importance of cell growth phase [4]. Previous studies have shown that some species and strains are more susceptible to transformation compared to others due to differences in the cell wall and membrane structure [5]. E. coli K12 derivatives are the most popular E. coli laboratory strains for transformation [6]. The composition of media for transformation efficiency is less important [7]. Transformation efficiency decreases linearly with an increase in the plasmid size [8]. Prior studies have shown that the transformation efficiency of E. coli DH5α strains is stimulated by several cations such as Mg2+, Mn2+, Rb2+, especially Ca2+. Some monovalent cations also have been reported to lead to concentration-dependent induction of transformation [9]. In this investigation, cell densities of around 107–109 cells/mL, (OD600 nm at 0.3–0.4 and 0.9-0.94), corresponding to mid-exponential phase and late growth phases were treated with different compounds of monovalent–halogen with or without divalent cations that resulted in a concentration–dependent competency of the cells. It was found that our improved competent cells can raise transformation efficiency about 102 times especially for ligation products and large DNA molecules (more than 10 kb).

MATERIALS AND METHODS

Bacteria, Plasmid, and Media

The Escherichia coli DH5α strain was employed for transformation. The plasmids were pBR322 (Ampr, Tetr) and ligation products of 5-12 kb. Cells were grown on Luria–Bertani (LB) and LB agar medium. The media were supplemented with appropriate antibiotics.

Chemical materials were purchased from Pharmacia Fine Chemicals Company (Uppsala, Sweden). The bacteria and plasmids were obtained from the National Institute of Genetic Engineering and Biotechnology (NIGEB; Tehran, Iran). All biochemical reagents were analytically pure. Plasmid DNA was extracted from E. coli by standard alkaline-lysis techniques [10].

Preparation of Competent Cell

10 μl of glycerol stock an E. coli DH5α was spread out on a fresh LB-agar plate and was grown overnight at 37 °C. A fresh single colony of the overnight culture was inoculated into 20-40 ml LB and was incubated overnight at 37°C by vigorous shaking. The overnight culture was diluted 1:20 with fresh LB and was shaken at 37°C until reaching the O.D600 of 0.94. The culture was ice-bathed for 20 min. The cells were harvested by centrifugation at 4000 rpm, at 4°C, for 10 min, and were gently re-suspended in 3 ml of ice-cold washing buffer I (Table 1) and were kept in ice for 10 min. The cells were harvested again by the same centrifugation. The supernatant was discarded, the pellet was gently resuspended in 3 ml of ice-cold washing buffer II (Table 1), and was kept in an ice bath for 10 min. The cells were collected by centrifugation at 3000 rpm, at 4°C, for 10 min. The supernatant was discarded and the pellet was gently resuspended in 1 ml of ice-cold storage buffer (Table 1). The cells were expected to become competent after overnight incubation (16 h) in the ice. All pipette tips used during the experiments were also chilled. The standard CaCl2 treatment for competent cell preparation was also used for comparative analysis of the results [11].

DNA Transformation

10 μl of plasmid DNA (5µg) was mixed with 100 μl of competent cell suspension and was incubated in an ice bath for 30 min. The mixture was heat-shocked in a water bath at 42̊C for 90-100 seconds and was rapidly transferred to an ice bath for 3-5 min. 400µl of LB medium was added and the mixture was incubated at 37̊C for 45-60 min.

Evaluation of Transformation Efficiency

25 μl of each sample was spread onto a selective LB agar plate containing the appropriate antibiotic. The plates were incubated at 37̊C for 18–20 h, and then the number of CFU (Colony Forming Units) formed per microgram of DNA was calculated.

Statistical Analysis

All the experimental data values were obtained from three independent experiments, and the results were presented as mean ± Standard Error of Mean (SEM). Statistical analyses were performed using SPSS software Ver.16.0 [12].

RESULTS AND DISCUSSION

Effect of Cell Growth Phase on Cell Competency

It has been previously reported that the state of the cell culture is important for transformation efficiency. Cell densities around 107–108 cells/mL (OD600 at 0.4), corresponding to the mid-exponential phase are considered as optimum for E. coli [13]. In this study, the E. coli DH5α cells were harvested at the OD600 range of 0.25-1.25 for competent cell preparation and transformation using the standard CaCl2 method. Competent cells were transformed with pBR322 plasmid DNA and were assayed for transformation and viability. Results showed that the OD600, of 0.35 and 0.95 yielded a higher frequency of transformation (Fig.1). Re-examination of cell culture resulted in 6×105 and 7×106 colony forming units per microgram of transforming DNA (CFU/μg) for cells harvested at the OD600, of 0.33 and 0.94, respectively. Most studies have investigated the effect of a limited range of OD600, usually around the OD600, of 0.45 on transformation efficiency [14]. The results of this research showed that transformation efficiency could also be enhanced at high cell densities (i.e., OD600 of 0.94).

This may be resulted from the cooperative transformation of the natural and artificial competency state of Escherichia coli. Some studies have shown that Escherichia coli cells harvested from stationary phase are able to uptake DNA from the environment [15]. Although, specific inducing signals for DNA uptake have not been known clearly, but nutritional state can be one of transformation regulatory machinery factors. Previous studies showed that, in laboratory conditions, usually natural competence is tightly regulated by nutritional shortages or adverse conditions [16]. Our results also showed that, competent cells made from bacterial cultures in stationary phase give larger colonies compared to the competent cells prepared from cultures with the OD600 of 0.4 (log phase) (Fig. 2). This property may also be attributed to 1) cell nutrition states (nutritional shortage state) which have a much tendency to over-eat resulting in nutrient and DNA uptake. Therefore, physiological state of the cells and probable natural transformation may contribute to high efficiency of our transformation, and 2) membrane of the E. coli which has hundreds of pores (adhesion zones) during rapid growth of the bacterial cells, as we postulated some of pores to be integrated to each other because of increasing number of pores at the end of log phase, as a result, they can absorb ligation product or large DNA.

Effect of Treatment Buffer on Competency

Composition and concentration of cations, such as Mg2+, Mn2+, Rb+, K+ and especially Ca2+  in treatment buffer are other important factors influencing the competency of bacterial cells [3]. The calcium chloride (CaCl2) method has been introduced as a popular efficient procedure, however, it is not enough effective, especially for the transformation of ligation product and large size DNA. In the present study, several improvements were employed to the treatment buffer (0.1M CaCl2) by replacing with different compositions and concentrations of monovalent and divalent cations (Table 1) to obtain efficient competent cells. Table 1 indicates transformation efficiency for different treatment buffers using pBR322 plasmid vector. Results can be summarized as follows: treatments II, III, and IV were 100-fold, 25-fold, and 10-fold more efficient than the standard CaCl2 method, respectively. For treatment I, efficiency was 40-fold less than CaCl2 method.

The hypotheses proposed for explanation of our results are as follows: 1) in this work, transformation efficiency was high when the cells were treated with storage or final treatment buffer containing monovalent cation and CaCl2. Prior studies showed that, presence and formation of poly-β-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes and divalent cations such as Ca2+, Mn2+, Sr2+, or Mg2+ are essential in genetic competence. The complexes are assumed to form channels or disintegrated lipid bilayer of the plasma membrane facilitating DNA transfer, and divalent cations increase penetration of the outer membrane and/or linking of DNA to PPi at the mouth of the channel complex by cross-linking phosphate groups of the complexes and DNA polymer. Huang and Reusch (1995) suggested critical selectivity of PHB/PPi for Ca2+ in the complexes; however, Das et al. [17] proposed that other divalent cations such as Sr2+, Ba2+, and Ca2+ could participate in the formation of complexes. They investigated the effect of monovalents, divalents and also the simultaneous effect of several cations and found high transformation efficiency for the competence-inducing buffer with three cations, Ca2+, Mn2+, and K2+ (10mM, 45mM, and 100mM, respectively).

Considering our results, we postulate that, calcium may participate in complex formation and potassium-mediated cell- membrane disintegration or DNA to PPi at complexes. We also assumed that the potassium with ionic radius bigger than calcium could produce large channel gate or cause cell membrane disintegration for transferring large size DNA. On the other hand, sufficient values of two cations with different roles induce the mentioned transformation process with no interference. Sufficient O/N incubation of cells at the last stage of preparation of competent cells is also effective. Sarkar et al. [18] indicated that CaCl2 mediates binding interaction between naked DNA plasmid with negatively charged lipopolysaccharide (LPS) molecules. Regarding transformation, they assumed transfer of DNA molecules across the least-barrier path at zones of adhesion, where the outer and inner cell membranes fuse with the pores in the cell wall, which are rich in negatively charged lipopolysaccharide (LPS) molecules. We believe that the potassium ions with a big radius can produce big shield around negatively charged DNA and help in transferring DNA molecules into the cell without much change in pH of cell medium.

In relation to the preference of washing buffer to the composition of KI/KCl over KCl, seemingly it is due to weak acidities of HI relative to HCl produced from KCl and KI breakup. HI is a weak acid and slightly changes the pH of the cell medium with the least effect on cell viability.

CONCLUSION

The results of this study indicated that competency and DNA transformation of E. coli could be further increased by employing a combination of monovalent and divalent cations for the preparation of competent cells from bacteria in the late log phase of cell growth. Therefore, modifications in the CaCl2 method presented in this study provide a more efficient E. coli transformation with great importance in genetic engineering and biotechnology.

REFERENCES

  1. Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Sciences of the United States of America. 1973;70:3240-4.
  2. Davis TO, Henderson I,Brehm JK, Minton NP. Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains. Journal of molecular microbiology and biotechnology. 2000;2:59-69.
  3. Aune TE, Aachmann FL. Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed. Applied microbiology and biotechnology. 2010;85:1301-1
  4. Aschar-Sobbi R, Abramov AY, Diao C, Kargacin ME, Kargacin GJ, French RJ, et al. High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach. J Fluoresc. 2008;18(5):859-66.
  5. Sambrook j, Russell DW. Molecular Cloning: A Laboratory Manual (3rd ed). Cold Spring Harbor Laboratory Press. 2001.
  6. Chen I, Dubnau D. DNA uptake during bacterial transformation. Nature reviews. Microbiology. 2004;2:241-9.
  7. Sambrook J, Russell DW. The inoue method for preparation and transformation of competent e. Coli: "ultra-competent" cells. CSH protocols. 2006.
  8. Aune TE, Aachmann FL. Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed. ApplMicrobiolBiotechnol. 2010;85(5):1301-13.
  9. PengD, Luo Y, Guo S, Zeng H, Ju S, Yu Z, et al. Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis. Journal of applied microbiology. 2009:106:1849-58.
  10. Turgeon N, Laflamme C, HoJ, Duchaine C. Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579. Journal of microbiological methods. 2006;67:543-8.
  11. Roychoudhury A, Basu S, Sengupta DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian journal of biochemistry & biophysics. 2009;46:395-400.
  12. Biswas GD, Sox T, Blackman E, Sparling PF. Factors affecting genetic transformation of Neisseria gonorrhoeae. Journal ofbacteriology. 1977;129:983-92.
  13. Liu J, Chang W, Pan L, Liu X, Su L, Zhang W, et al. An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments. Indian J Microbiol. 2018;58(4):448-56.
  14. Wu N, Matand K, Kebede B, Acquaah G, Williams S. Enhancing DNA electrotransformation efficiency in Escherichia Coli DH10B electrocomponent cells. Electronic Journal of Biotechnology. 2010;13:1-9.
  15. Tsen SD, Fang SS, Chen MJ, Chien JY, Lee CC, Tsen DH. Natural plasmid transformation in Escherichia coli. Journal of biomedical science. 2002;9:246-52.
  16. Solomon JM, Grossman AD. Who's competent and when: regulation of natural genetic competence in bacteria. Trends in genetics : TIG. 1996;12:150-5.
  17. Das S, Lengweiler UD, Seebach D, Reusch RN. Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proceedings of the National Academy of Sciences of the United States of America. 1997;94:9075-9.
  18. Sarkar S, Chaudhuri S,  Basu T. Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol. Current Science. 2002;83:1376-1380.

 

 

 

Figure 1. Transformation of competent E. coli DH5α cells derives from various stages of growth.

 

Figure 2. Comparison of colony size of E. coli transformants obtained from cultures with OD600 of 0.33 (Left) and 0.94 (Right).

Table 1. A comparison of different treatment composition and concentration for competency experiment.

 

Treatment

Washing Buffer I

Washing Buffer II

Storage  Buffer1

Efficiency of

transformation

(CFU/µg DNA)

 

Component

Concentration

(mM)

Component

Concentration

(mM)

Component

Concentration

(mM)

 

Treatment I

 

KCl

KI

150

100

KCl

KI

NH4Cl

100

50

50

KCl

 

150

 

(2.3±1.1)×105

 

Treatment II

 

KCl

KI

150

100

KCl

KI

80

80

KCl

CaCl2

80

80

(8.2±2.1)×108

 

Treatment III

KCl

150

KCl

150

KCl

CaCl2

80

80

(2±1.8)×108

 

Treatment IV

 

KCl

MgCl2

150

100

KCl

MgCl2

150

100

CaCl2

100

(8.3±4.9)×107

 

Treatment V

CaCl2

100

CaCl2

100

CaCl2

100

(8.1±5.6)×106

1 The storage buffer contains the same volume of ice-cold 40% glycerol and cation(s).

scan0008

Figure 2. Comparison of colony size of E. coli transformants obtained from cultures with OD600 of 0.33 (Left) and 0.94 (Right).

Table 1. A comparison of different treatment composition and concentration for competency experiment.

 

Treatment

Washing Buffer I

Washing Buffer II

Storage  Buffer1

Efficiency of

transformation

(CFU/µg DNA)

 

Component

Concentration

(mM)

Component

Concentration

(mM)

Component

Concentration

(mM)

 

Treatment I

 

KCl

KI

150

100

KCl

KI

NH4Cl

100

50

50

KCl

 

150

 

(2.3±1.1)×105

 

Treatment II

 

KCl

KI

150

100

KCl

KI

80

80

KCl

CaCl2

80

80

(8.2±2.1)×108

 

Treatment III

KCl

150

KCl

150

KCl

CaCl2

80

80

(2±1.8)×108

 

Treatment IV

 

KCl

MgCl2

150

100

KCl

MgCl2

150

100

CaCl2

100

(8.3±4.9)×107

 

Treatment V

CaCl2

100

CaCl2

100

CaCl2

100

(8.1±5.6)×106

1 The storage buffer contains the same volume of ice-cold 40% glycerol and cation(s).

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