Foreign DNA can be introduced into a cell through the transformation process. The transformation of bacteria with plasmids is not only important for bacterial studies, but can also be used for gene expression studies in mammalian cells. Most plasmids are of bacterial origin and contain both a bacterial origin of replication and an antibiotic resistance gene that can be used as a selectable marker.
Genetic modifications can be made to different strains of bacteria to make them more susceptible to transformation. Such modifications will preserve the plasmid without rearranging the plasmid DNA. Certain treatments have been shown to increase the transformation efficiency of bacteria. These treatments make them more susceptible to chemical or electrical conversions, creating what are known as "competent cells."
change in practice
The ability to selectively isolate, amplify, and propagate selected DNA sequences is of fundamental importance in biology. It has much more practical and everyday uses than the cloning we read about, e.g. Dolly the Sheep Cloning allows us to amplify DNA sequences of interest and takes advantage of 2 classic properties of bacteria, plasmids and restriction endonuclease.
Plasmids can be found in bacteria, but they can replicate independently of chromosomal DNA, are double-stranded, and are generally circular. Plasmids are ideal for cloning because they can accept foreign DNA to produce a chimera containing both their own DNA and the DNA selected for amplification; the plasmid is now said to be recombinant. They also possess a restriction endonuclease, an origin of replication, and a selection marker, e.g. Resistance to kanamycin or ampicillin.
Once recombinant, the plasmid can turn back into the bacterium. This is usually achieved by making the cells competent, e.g. B. by heat shock to weaken the membrane and allow bacteria to take up the plasmid.
After transformation, the bacteria can be screened or selected for plasmid/vector uptake, which is generally accomplished by plating the bacterial broth onto agar. For example, let's say the gene of interest is thisIL-18Promoter, this can be inserted into the LacZ gene on the plasmid encoding B-galactosidase. Galactosidase B can digest X-Gal to produce blue colonies. However, if the gene of interest is successfully inserted, the expression of B-galactosidase will be disrupted and the colonies will appear white on the agar plate.
Equipment/materials needed for the transformation
*These may vary depending on the transformation protocol
- competent cells
- Agar LB
- social media
- Antibiotic (Kanamycin/Ampicillin)
- Cultural plate
- Incubator with shaker at 37°C
- Incubation cabinet at 37°C
- Water bath at 37°C
Fabrication of Chemically Competent Cells (Example Protocol 1)
- Escherichia colithe DH5 alpha strain was used to amplify the plasmid DNA
- cellsthey were made competent by the calcium chloride method.
- Untransformed DH5 alpha was plated on LB agar (Luria Bertani) (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) supplemented with 15 g/l l agar and left overnight incubated at 37 °C.
- Then a well-defined DH5 alpha colony was picked from the LB agar plate and cultured overnight in 5 ml LB (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) with shaking at 200 rpm.
- Next, 1 ml of the overnight culture was diluted to 150 ml with LB and grown to an OD600 of 0.4-0.6.
- The cells were collected by centrifugation at 800 rpmGrammfor 10 min at 4°C
- Resuspend in 10 mL of sterile, ice-cold 0.1 M CaCl2 and incubate on ice for 5 min.
- After recentrifugation, the cells were resuspended in 2 ml 0.1 M CaCl 2 and 70 µl DMSO was added.
- The cells were reincubated on ice for 15 min. Aliquots of 200 µl were stored at -80°C.
Transformation Protocol for E.Coli Competent Cells (Example Protocol 1)
- Frozen competent cells were thawed on ice.
- 100 µl aliquots of the bacteria were thawed.
- Next, 2 µl of the desired plasmid was added and the cell/plasmid mixture was incubated on ice for 30 minutes.
- Plasmid uptake was induced by applying heat to the cells at 42°C for 40 seconds followed by a 2 minute recovery on ice.
- Next, 500 µl of LB was added and the cells were incubated for 1 hour with gentle shaking (220 rpm) to allow expression of the antibiotic resistance gene.
- 100 µl aliquots of cells were plated onto LB agar plates containing ampicillin (100 x g/ml) to select for transformants harboring the plasmids.
- The dishes were inverted and incubated overnight at 37°C.
Preparation of Competent E. coli Cells (Example Protocol 2)
- A single colony ofcoliXL1 was inoculated into 5 ml of LB medium and cultured overnight at 37°C on a shaker.
- The 5 ml culture was added to 100 ml of LB medium in a 250 ml sterile flask and grown on a shaker at 37°C until the absorbance at 600 nm (OD600 nm) reached 0.6 AU.
- The culture was transferred to 50 mL tubes and chilled on ice for 10 min.
- The samples were centrifuged at 1700 RCF for 10 min and the supernatant was removed.
- The bacterial cell pellet was used to prepare glycerol stocks by resuspending the pellet in 2 mL of 40% glycerol in distilled H 2 O.
- 100 µl were aliquoted into 1.5 ml tubes and snap frozen using liquid nitrogen and stored at -80°C.
- Before transformation with plasmid DNA, a glycerol stock solution ofcoliXL1 was removed from storage at -80°C and made competent by pelleting the cells by centrifugation at 2700 RCF for 5 min.
- The supernatant was removed and the pellet resuspended in 10 µl TfbI buffer (30 mM potassium acetate, 100 mM rubidium chloride, 10 mM calcium chloride, 50 mM manganese chloride, 15% (v/v) glycerol), pH 5.8 with acetic acid, sterile-filtered) and incubated on ice for 15 min.
- The suspension was centrifuged at 2700 RCF for 5 min and the supernatant removed and the pellet suspended in 10 μl TfbII buffer (10 mM MOPS, 75 mM calcium chloride, 10 mM rubidium chloride, 15% (v/v) glycerol, pH 6.5 with NaOH, sterile filtered).
Competent E. coli Transformation (Example Protocol 2)
- CompetentcoliXL1 cells (10 µl) prepared as above (Preparation) Competent E. coli cells (sample protocol 2) were incubated with 2 µl of plasmid DNA and placed on ice for 30 min with occasional mixing.
- Samples were heat shocked in a 37°C water bath for 2 min and then transferred to ice.
- The cells were then added to 1 ml of LB broth and placed on a shaker at 37°C for 60 minutes, followed by centrifugation at 3000 RCF for 3 minutes.
- The supernatant was removed leaving 100 µl and the pellet was resuspended in the remaining supernatant.
- An aliquot (100 µl) of the sample was streaked onto an LB agar plate containing 30 µg/ml kanamycin and incubated overnight at 37°C.
After overnight expansion, the bacterial cells can be pelleted by centrifugation and the plasmid purified therefromE coli.The plasmid DNA can then be quantitated using the NanoDrop 800 Multi-Sample Microvolume UV-Vis Spectrophotometer or similar devices. DNA can be stored at -20 °C for short term or at -80 °C for long term.
1. Temperature–Temperature plays a very important role in the transformation and can greatly affect the efficiency of your experiment. The right temperature is critical at three stages: thawing, incubation of DNA with cells on ice, and during heat shock.
- Thawing-Cells thaw best on ice. Cells can be thawed by hand, but heating above 0°C reduces efficiency. Once the last trace of ice has disappeared, the DNA must be added.
- Incubating DNA with Cells on Ice –Ideally, incubate on ice for 30 minutes. Expect a 2x loss in TE for every 10 minutes you shorten this step.
- heat stroke -The combination of chemical ions and rapid changes in temperature (i.e. "heat shock") likely changes the permeability of the bacterial cell wall and membrane, allowing DNA molecules to enter the cell. For best results, keep the cell suspension ice cold before and after the heat shock. Before the heat shock, use a thermometer to confirm that the water bath reaches 42 °C.
2. Resuspension of bacteria –If the cells are not completely resuspended, the plasmid will not come into contact with most bacterial cells. The cells should be resuspended by pipetting up and down until no clumps are visible. Also try to prevent the cells from heating up during resuspension, this will affect their heat shock later. It is good not to heat the cells with your hands by holding the tube at the top and not at the bottom where the cells are.
3. Sin Transformants –The most obvious first step would be to verify that you are plating on an LB agar plate containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic on the plate. You should also add a positive control to ensure your transformation procedure is working. It's understandable that too little plasmid DNA decreases transformation efficiency, but did you know that adding too much plasmid can also affect your results? Sometimes less is more. It may seem counterintuitive, but you often get higher transformation efficiencies with less DNA, especially when using highly competent cells. If you have used 100-1000 ng of total DNA in a ligation, you will often get more colonies if you use 1 µL of a 1:5 or 1:10 dilution instead of 1 µL directly.
4. Growing time–Growth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2x loss in TE for every 15 minutes you shorten this step. Incubation with shaking or rotating the tube gives a 2-fold higher ET.
March 15, 2021 alter macferrari