Relative variability in transformational efficiency of CaCl 2 , MgCl 2 , Tris-HCl, and Tris EDTA in E coli and Agrobacterium tumefaciens

Bacterial transformation is an inevitable tool for gene manipulation and plant transformation. Ca2+ divalent cations are indispensable for the induction of competency in E coli transformation. Different types of ions have been reported to induce and enhance transformation in E coli with varied efficiency. In the current study we investigated the effect of CaCl2, MgCl2, Tris-HCl, Tris EDTA, NaOAc and NH4OAc in Escherichia coli and Agrobacterium tumefaciens. The results indicate that the effect of chemicals on E coli and agrobacterium may depend on the electrochemical physiology of the cell and the membrane characteristics. It is reported in the current study that the efficiency of mediator chemicals are appreciably varying when related to the two samples.


Introduction
Transformation is the process of uptake of exogenous DNA or plasmid by competent E coli cells.
This involves two stages, firstly the E coli cells become competent to intake DNA from the environment and secondly the entry of DNA across the bacterial membrane. After the first artificial transformation of E coli using CaCl 2 1 gram negative bacteria were artificially transformed using plasmids 2, 3 , but it was Hanahan 4, 5 who established that cation induced competency in E coli was the criterion for transformation.
Ability of CaCl 2 to induce competency in bacterial cells for transformation has proven to be very efficient and is the most commonly used chemical method. Competency is the physiological condition of the cell wherein the electrostatic repulsive forces between DNA and membrane is reduced 6 . It is suggested that cations form coordination complex with DNA and lipopolysaccharides (LPS) 7-9 . In addition DMSO, DTT, mercaptoethanol and 10% ethanol have enhancing effects on transformation efficiency 4 .
There is a direct impact of efficiency of transformation in cloning works involving genomic DNA, cDNA or EST library preparation. Efficiency in transformation is the number of CFU produced per microgram of plasmid used. It would be appropriate to have the best conditions to achieve efficient transformation. As a consequence, this study was done to evaluate the potential of various chemicals like Tris HCl, Tris EDTA, NaOAc and NH 4 OAc alongside CaCl 2 and MgCl 2 in E coli and Agrobacterium tumefaciens transformation. Transformation of E coli and A tumefaciens were carried out with plasmids pUC18 and pCAMBIA 2300 respectively.

Materials and methods
E coli strain Top 10, A tumefaciens strain LBA4404 were the bacterial strains used.

Antibiotic preparation
Rifampicin was prepared by dissolving 10 mg of the antibiotic in 1ml methanol.
Kanamycin was prepared by dissolving 50 mg of the antibiotic in 1ml sterile water.
Streptomycin was prepared by dissolving 50 mg of the antibiotic in 1ml sterile water.
Competent cell preparation E. coli and A tumefaciens were cultured in LB and YEP broth with ampicillin 100 mg/l and rifampicin 10 mg/l respectively. 1% inoculum was used for competent cell preparation. Cells were harvested at the physiological active stage (O.D 0.3-0.5 at A 600 ) and pellet made at 5000rpm for 5 minutes at 4˚C. Resuspension of pellet was done in 1 ml of ice cold filter sterilized competent solution (various concentrations of CaCl 2 , MgCl 2 , Tris-EDTA, Tris-HCl, YEP, NaOAc, NH 4 OAc). The cells were placed on ice for 30 minutes and pelleted out at 5000rpm for 5 minutes at 4˚C. The pellet was re-suspended in 80µl of respective competent solution and 20µl of 98% glycerol and was stored in -70 0 C till transformation.

Cell viability assay
Cell viability test for E coli and A tumefaciens competent cells were performed alongside transformation without plasmids. 100 µl of culture at 10 -8 dilution was cultured on petriplate without selection. The number of colonies was counted to calculate the colony forming units. All experiments were performed in triplicates.
E coli transformation E coli competent cells were added with 10 ng of plasmid pUC18 and treated with ice for 30 minutes. The cells were given a heat shock for 90 seconds at 42 o C and placed on ice for 5 minutes. The cells were added with 900µl LB broth and incubated in shaker at 37 O C for 1 hour. 100µl of the culture was spread plated on LB medium with ampicillin (100µg/ml). The plates were incubated in a static incubator overnight at 37 O C. All experiments were performed in triplicates.

Agrobacterium transformation
The Agrobacterium competent cells along with 1µg of plasmid pCAMBIA 2300 were treated with ice for 10 minutes and in -70˚C for 1 hour. The cells were thawed on ice for 5 minutes and incubated at 37 0 C for 10 minutes. The cells were placed on ice for 5 minutes and 900µl of YEP broth was added and incubated in a shaker for 3 hours at 28˚C. The culture was pelleted and resuspended in 100µl of YEP broth and spread plated on YEP media containing rifampicin (10µg/ml) and kanamycin (50µg/ml). Theses plates were incubated in static incubator at 28˚C for 2 days. All experiments were performed in triplicates.

Colony PCR
Three colonies from transformed plates were randomly inoculated in 41µl of sterile water and the PCR master mix (nptII primers 1 µl each 5' TCAGAAGAACTCGTCAAGAAG 3' and 5' ATGGGGATTGAACAAGATG 3', 10mM dNTPs 1µl, 10X taq buffer 5 µl, Taq polymerase 1unit ) was added. The samples were amplified with the given condition, an initial hold at 94 o C for 5minutes, 35 cycles of 94 o C -40 seconds, 58 o C -40 seconds, 72 o C -40 seconds and final extension of 2 minutes at 72 o C. The amplified product was checked using 1% agarose gel.

Results
The transformants in Agrobacterium for pCAMBIA was confirmed by colony PCR (fig. 7).

Effect of CaCl 2 in E coli and Agrobacterium transformation
The efficiency of transformation in E coli was maximum at 100mM concentration with 1.31 X 10 7 CFU. The efficiency was 1.37 X10 6 CFU in 10 mM with cell viability 1.25 X10 10 CFU. The transformation efficiency increased with increase in the concentration of CaCl 2 and had maximum at 100 mM ( fig. 3). The efficiency in agrobacterium transformation was 2.7 X10 3 CFU with a cell viability of 1.35 X10 10 at 10mM concentration ( fig. 2). The efficiency of transformation decreased with increase in the concentration ( fig. 4). Further, CaCl 2 induced competency produced 38% of the transformants in E coli ( fig. 5) and only 16% in agrobacterium ( fig. 6).

Effect of Tris EDTA and MgCl 2 in E coli and Agrobacterium transformation
Tris EDTA and MgCl 2 produced about 7.0 X10 5 CFU and 1.08 X10 6 CFU in E coli at 10mM concentration and 2.9 X10 3 CFU and 2.1 X10 3 CFU in agrobacterium. As the concentration increased the efficiency reduced in both the bacteria. Tris EDTA produced 19% and 20% transformants while MgCl 2 produced 7% and 30% transformation in agrobacterium and E coli respectively. Tris EDTA at 10mM had positive effect on agrobacterium transformation but repressed E coli transfor-mation at higher concentration.
Effect of NaOAc and NH 4 OAc in E coli and Agrobacterium transformation NaOAc and NH 4 OAc showed lower transformation efficiency with E coli and agrobacterium. They produced 2.7 X10 5 CFU and 2.0 X10 4 CFU in E coli and 4.0 X10 2 CFU and 1.2 X 10 3 CFU in agrobacterium respectively.

Discussion
In principle the entry of DNA in to the bacteria is based on the transmembrane electrochemical gradient. The requirement of Ca 2+ ions at 0 o C, and independent heat shock in Ca 2+ induced bacterial transformation is established. DNA in solution bind strongly to CaCl 2+ and with increased extracellular concentration of Ca 2+ the DNA uptake is induced 10 . In our study, Ca 2+ ions had varied effect on the two bacteria.
The varied effect of tris-HCl on E coli and agrobacterium could be due to difference in the membrane characteristics of the individual bacteria.
This may be due to EDTA which has two types of activity on the cell membrane. At low concentration it leaches out the LPS in bacteria enhancing transformation and at higher concentration deforms the cell by leaching out up to 40% LPS, outer membrane proteins, OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine 15 . MgCl 2 was preferred by E coli rather than agrobacterium. It is known that MgCl 2 is required for the stabilization of LPS present on the outer membrane of E coli. Tris EDTA also chelates Mg 2+ ions required for stabilization of the LPS on the outer membrane of the bacteria 14, 15 .
This confirms the toxic effect of NH 4 ions on cell and inhibiting activity of Na + ions in DNA LPS binding 16 .

Conclusion
Ca 2+ ions at 100mM show highest efficiency in E coli and Tris-HCl at 100mM show highest efficiency in agrobacterium. Based on our study, the efficiency of the chemicals are of the order CaCl 2 > MgCl 2 > tris EDTA > Tris-HCl and Tris-HCl > Tris EDTA > CaCl 2 > MgCl 2 in E coli and Agrobacterium tumefaciens respectively. The efficiency of transformation in bacteria may also be influenced by the membrane characteristics which need further scientific evidence.