Journal of Research in Biology
Transformation efficiency on E. coli in response to different bivalent salts
Keywords:
Transformation efficiency, E. coli, Bivalent salt.
ABSTRACT:
Bacterial transformation is the process of up taking foreign DNA molecules by bacterial cells. Bacteria, which are able to up take such foreign DNA are called “competent” cell. Foreign DNA carrying antibiotic resistance gene can be expressed in the bacteria and transforming the bacteria from antibiotic sensitive to antibiotic resistance. Such transformed bacteria can easily be isolated. The efficiency of the transformation plays a critical role in recombinant DNA technology. Several methods have been developed to improve the transformation efficiency. In our work we have studied the transformation efficiency on strains of E. coli and in response to different bivalents salts at different concentrations. All the experiments revealed that transformation efficiency was highest on E. coli DH5α strain in 50 mM CaCl2 concentration. Comparing to the other E. coli and bivalents salts concentration, 50 mM CaCl2, resulted in much faster growth of transformed bacteria. Thus, our results suggest that E. coli DH5α strain and CaCl2 can be used as an effective agent for the transformation technique.
704-710 | JRB | 2012 | Vol 2 | No 8
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Authors:
Sahanawaz Alam SK and Panigrahi AK.
Institution:
Fisheries and Aquaculture Extension laboratory,
Department of Zoology, University of Kalyani, Kalyani, West Bengal. India.
Corresponding author:
Sahanawaz Alam Sk.
Email:
sahanawaz11@gmail.com
Web Address:
http://jresearchbiology.com/documents/RA0295.pdf.
Dates:
Received: 27 Oct 2012 Accepted: 05 Nov 2012 Published: 11 Dec 2012
Article Citation:
Sahanawaz Alam Sk and Panigrahi AK.
Transformation efficiency on E. coli in response to different bivalent salts.
Journal of Research in Biology (2012) 2(8): 704-710
Journal of Research in Biology
An International Scientific Research Journal
Original Research
INTRODUCTION
Transformation is defined as the uptake and expression of foreign DNA by cells. Transformation of bacteria involves DNA binding to all cell surface followed by uptake across the wall - membrane complex into the cytoplasm. Bacterial transformation occurs naturally in many bacterial genus such as Micrococcus, E. coli, Haemophilus and Bacillus (Michod et al., 1988) all these organisms have proteins on their exterior surface whose function is to bind to DNA in their environment and transport it into the cell. However, it is still a rare event for most bacteria to naturally take up DNA from the environment. By subjecting bacteria to certain artificial conditions, many of them become able to take up free DNA and the cells in such state are referred to as competent.
In early seventies, the discoveries of transferring the phage (Mandel and Higa, 1970) and plasmid (Cohen et al., 1972) DNAs into Escherichia coli cells following a competence induction process involving treatment with calcium chloride, set the stage for molecular cloning of recombinant DNAs. The technique of DNA transformation has then become important in virtually all aspects of molecular genetics. E. coli has developed into a universal host organism both for molecular cloning of DNA and for a diverse set of assays involving cloned genes. In E. coli the competence can be developed by suspending the cells in ice-cold CaCl2 and then subjecting to a brief heat shock at 42°C (Cohen et al., 1972; Mandel and Higa, 1970). Even after generation of competence, the technique of E. coli transformation is highly inefficient; the vast majority of DNA molecules added does not enter any cell, and in turn the vast majority of bacterial cell receives no DNA. With the advancement of time, the original method of competence induction by CaCl2 treatment has gradually been improved to different high-transformation-efficiency protocols, which employ magnesium ions, manganese ions (Hanahan, 1983; Hanahan et al., 1991). Besides the chemical methods, physical treatment like electroshock with a brief pulse of high voltage electricity has been shown to be applicable to competence induction in E. coli and other bacteria (Neumann et al., 1982).
Even after all such developments, the exact mechanism of CaCl2 mediated artificial transformation is still largely obscure. It is believed that the CaCl2 helps in DNA adsorption of the competent cell surface and the heat-shock step facilitates for penetration of the adsorbed DNA into the cell cytosol. Earlier finding (Sarkar et al., 2002a) shows that during artificial transformation of E. coli, the naked DNA is bound to the lipopolysaccharide (LPS) receptor molecules on the competent to form surface; the divalent cation Ca2+ is suggested to form coordination complexes with the two negatively charged macromolecules - DNA and LPS. In this study, our primary motivation is to investigate the mechanism of DNA entry into the cell cytoplasm during artificial transformation. Here, it has been shown that heat shock step of the standard transformation procedure heavily depolarizes the membrane of CaCl2-treated component cells. So, we have used different type of bivalent salts for transformation. The aim of this experimentation is to study the efficiency of transformation in respect to different bivalent salts.
MATERIALS AND METHODS
Bacterial Strain – E. Coli DH5.
Plasmid – pUC-19
Media for bacterial growth
LB medium
Bacto-tryptone-10 gm/L, Bacto-yeast-5 gm/L, Nacl-10 gm/L, pH was adjusted to 7.0-7.4 with NaOH (10 N) and was autoclaved to sterilize. The autoclaved medium was cooled to 55°C and ampicillin was added (final concentration 50 mg/ml). For LB-agar plates, 1.7% Bactoagar (17 gm/L) was added prior to autoclaving.
Salts
CaCl2, CoCl2, MgCl2, NaCl, NH4FeSO4, MgSO4
was used in 25 mM, 30 mM, 50 mM and 80 mM concentration. All salts were added as solids, always kept and used in cold.
Methods
Preparation of E. coli DH5a competent cell
The following simple procedure is a variation of that of (Cohen et al., 1972) and is frequently used to prepare batches of competent bacteria that yield, 5x106 to 2x107 transformed Colonies per microgram of super coiled plasmid DNA. The standardization of procedure is established in our research laboratory.
Bacterial transformation
According to (Zhiming et al., 2005) and we modify the Transformation process in our laboratory.
Plasmid transformation and antibiotic selection Calcium chloride treatment of bacterial cells produces competent cells that will take up DNA following a heat shock step. DNA molecules, i.e. plasmid, which are introduced by this method, will then be replicated in the bacterial host cells. To aid the bacterial cells’ recovery, the cells are incubated briefly with non-selective growth medium following the heat shock treatment. However, due to the low percentage of bacterial cells that have been transformed with the
plasmid and the potential for the plasmid not to propagate itself in all daughter cells, it is necessary to select for bacterial cells that contain the plasmid. This is commonly performed using antibiotic selection.
E. coli strain such as DH5 is sensitive to common antibiotics such as ampicillin. Plasmids used for the cloning and manipulating of DNA have been engineered therefore to harbor genes for antibiotic resistance too. Only bacteria that posses the plasmid DNA will have the ability to metabolize ampicillin and form colonies. Bacterial cells containing plasmid DNA has been selected following this technique.
Protocol of Bacterial transformation
RESULTS AND DISCUSSION
Using cells in early log phase of growth is an important factor for preparation of competent cells. We have chosen the optimum optical density at 600 nm (OD600) for the preparation of competent cells using strain DH5 where it was 0.25-0.30. We have studied the effects of different bivalent salts on the transformation efficiency of DH5 using the growth medium LB as the most suitable one.
Calculation of transformation efficiency
Transformation efficiency is defined as the number of colony forming units (cfu) produced by 1 mg of plasmid DNA, and each measured by performing a control transformation reaction using a known quantity of DNA, then calculating the number of cfu formed per mg of DNA.
Equation for transformation efficiency
Transformant cfu = No. of bacteria colonies × dilution ratio × original transformation volume / plated volume.
Transformation efficiency = Transformant cfu/plasmid DNA added (mg)
The plasmid DNA was added 1.4 mg,
The following table shows the average transformation efficiency to that of the salts used.
Transformation efficiency is very important in molecular cloning experiments, and can be affected by many factors. Takahashi have reported a simple method of plasmid transformation of E. coli by rapid freezing
(Takahashi et al., 1992). The most important being that the bacterial cells must in their early logarithmic growth period, Ryu and other authors have pointed out the importance of the early log phase for transformation (Ryu and Hartin, 1990). Bacteria that are able to take up DNA are called "competent" and competency can be induced by treatment with calcium chloride in the early log phase of growth. Bacteria that are able to take up DNA are called “competent” and competency can be induced by treatment with bivalent chloride and sulphate salts in the early log phase of growth. The bacterial cell membrane is permeable to chloride and sulphate ions, but is non permeable to Calcium ions. As the Chloride ions enter the cells, water molecules accompany the charged particle. This influx of water causes, the cells to swell and is necessary for the uptake of DNA, The exact mechanism of this uptake is unknown. Our experiments have shown that E. Coli DH5, the optimum OD600 is 0.25-0.30, is optimum for bacterial transformation efficiency. For competent cells preparation it would be routinely cultured to OD600 = 0.25-0.03.
Salt has an impact on transformation efficiency, in response to bivalent salts like CaCl2, CoCl2, MgCl2, NaCl, NH4FeSO4, MgSo4, the transformation efficiency was found to be highest in CaCl2 as depicted by figure-1.
According to (Brian and Heller, 1996) shows that CaCl2 75 mM concentration is optimum in TB solution. Another important factor is the concentration of divalent salt like CaCl2, CoCl2, MgCl2, NaCl, NH4FeSO4, MgSO4, although 25-80 mM salt concentration was used but 50 mM was shown to be optimum for all the cases as depicted by figure-2.
CONCLUSION
The present study shows that bacterial transformation process is associated with the bivalent salt and also associated with bacterial optimum growth phase and also salt concentration. We observed that Escherichia coli DH5 strain bacterial transformation processes were optimum on OD600 = 0.25-0.03 and the transformation efficiency was found to be highest in CaCl2 at 50 mM concentration. CaCl2 a bivalent salt can be used as an effective agent for the transformation technique.
REFERENCES
Brian P and Heller Mk. 1996. High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Research 24(3):536-537.
Hanahan D, Jessee J, Bloom FR. 1991. Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology. 204:63-113.
Hanahan D. 1983. Studies on transformation of E.coli with plasmids. Journal of Molecular Biology. 166:557-580.
Mandel M and Higa A. 1970. Calcium dependent bacteriophage DNA infection. Journal of Molecular Biology. 53(1):159-162.
Michod RM, Wojciechowski M, Hoelzer M. 1988. DNA repair and the evolution of transformation in the bacterium Bacillus subtilis. Genetics (1):118:31-39.
Neumann E, Schaefer-Ridder M, Wang W, Hofschneider PH. 1982. Gene transfer in mouse lyoma cells by electroporation in high electric fields. European Molecular Biology Organization. Journal 1(7):841-845.
Ryu J and Hartin RJ. 1990. Quick transformation in Salmonella typhimurium LT2. Biotechniques 8(1):43-44.
Stanley N. Cohen, Annie C. Y. Chang, and Leslie Hsu. 1972. Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA. Proceedings of the National Academy of Sciences of the United States of America. 69(8):2110-2114
Sarkar S, Choudhuri S, Basu T. 2002a. Ethanol-induced enhancement of the transformation of Escherichia.coli by plasmid DNA. Ind. Indian Journal of Biotechnology.1 (2):209-211.
Takahashi R, Valeika SR, Glass KW. 1992. A simple method of plasmid transformation of E.coli by rapid freezing. Biotechniques.13(5):711-715.
Zhiming Tu, Guangyuan He, Kexiu X. Li., Mingjie J. Chen, Junli Chang, Ling Chen, Qing Yao, Dongping P. Liu. 2005. An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electronic Journal of Biotechnology 8(1):114-120.
Alam and Panigrahi, 2012
705 Journal of Research in Biology (2012) 2(8): 704-710
Alam and Panigrahi, 2012
Journal of Research in Biology (2012) 2(8): 704-710 706
b. Magnesium sulphate (MgS04)
Transformation efficiency
a. Calcium chloride (CaCl2)
Transformation efficiency
c. Magnesium Chloride (MgCl2)
d. Ammonium-Ferrous sulphate (NH4FeS04)
Transformation efficiency
Transformation efficiency
e. Sodium Chloride (NaCl)
f. Cobalts Chloride (CoCl2)
Figure 1 In all the transformation process (a, b, c, d, e, f) 50 mM
concentration is the highest for with above bivalents salt.
Transformation efficiency
Transformation efficiency
Transformation efficiency
Concentration of salt
Concentration of salt
Concentration of salt
Concentration of salt
Concentration of salt
Concentration of salt
Alam and Panigrahi, 2012
707 Journal of Research in Biology (2012) 2(8): 704-710
Salt |
Concentration (mM) |
Average Transformation Efficiency (cfu/mg) |
a. Calcium chloride (CaCl2) |
25 |
0.590 |
30 |
4.675 |
|
50 |
23.98 |
|
80 |
0.980 |
|
b. Magnesium sulphate(MgSo4) |
25 |
0.110 |
30 |
2.367 |
|
50 |
3.037 |
|
80 |
0.310 |
|
c. Magnesium Chloride(MgCl2) |
25 |
0.073 |
30 |
0.344 |
|
50 |
0.699 |
|
80 |
0.000 |
|
d. Ammonium-Ferrous-sulphate (NH4FeS04) |
25 |
0.607 |
30 |
0.774 |
|
50 |
1.424 |
|
80 |
0.052 |
|
e. Sodium Chloride (NaCl) |
25 |
0.178 |
30 |
0.658 |
|
50 |
1.543 |
|
80 |
0.000 |
|
f .Cobalts Chloride (CoCl2) |
25 |
0.756 |
30 |
1.035 |
|
50 |
1.601 |
|
80 |
0.000 |
Table 1 shows that transformation rate is high in 50 mM concentration of each bivalent salts.
Alam and Panigrahi, 2012
Journal of Research in Biology (2012) 2(8): 704-710 708
Salt |
Average of Transformation Efficiency |
a. NaCl |
0.7932 |
b. MgSo4 |
2.2136 |
c. CoCl2 |
1.1306 |
d. CaCl2 |
9.7483 |
e. MgCl2 |
0.4172 |
f. NH4FeSO4 |
0.9350 |
Figure 2 shows that the transformation efficiency is highest in using CaCl2 (d) as a bivalent salt
according to above table.
Transformation efficiency
different types of salt
Alam and Panigrahi, 2012
709 Journal of Research in Biology (2012) 2(8): 704-710
Alam and Panigrahi, 2012
Journal of Research in Biology (2012) 2(8): 704-710 710
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