Transformation
Created: Sep 2021
Objective
Transferring DNA from a certain species to another (genetically different) species in order to induce genetic changes is called transformation. Adding genetic traits (abilities) not originally possessed by bacteria such as E. coli etc. through incorporating plasmid DNA into them, thus transforming the cells, is an important technology in genetic engineering.
In this experiment, a plasmid DNA carrying an antibiotic resistance gene is introduced into E. coli in order to create an E. coli recombinant that has acquired the antibiotic resistance trait.
In general, plasmids are not incorporated into E. coli in nature. Therefore, it is necessary to change the surface state of E. coli cells by artificial processing so that the plasmid can be introduced. This is called competence, and there are various chemical and physical methods of achieving it. The cells that have been made competent and into which the plasmid can be introduced are called recipient cells (competent cells).
In this experiment, E. coli DHSα strain is made competent by a simple calcium chloride method, and plasmid DNA (pUC19) containing the ampicillin resistance gene is introduced into the obtained cells. As the result, E. coli which were originally sensitive to ampicillin become resistant to it. After the bacteria have been cultivated overnight, E. coli into which the plasmid has been incorporated form colonies. The number of colonies represents the number of bacteria in which transformation occurred. In addition, the viable count is also determined.
The newly formed colonies are transferred to liquid medium, cultured, and used in the next experiment (plasmid isolation).
The objective of this experiment is to induce transformation in E. coli cells, making them immune to ampicillin. Moreover, we will calculate the transformation rate and the percentage of bacteria that underwent transformation.
Method
Equipment
• Micropipettes
• Micropipette tips
• Tubes
• Clean bench
• Centrifuge
• Gas burner
• Spreader
• Incubator
• High temperature water tank
Reagents
• E. coli DH5α strain
• pUC19 (plasmid)
• 0.1 M CaCl2
• SOC medium
• 0.9% NaCl
• LB medium agar plate
• LB medium agar plate containing 100 mg/L ampicillin
• Liquid LB medium containing 100 mg/L ampicillin
Precautions
Do not discard the bacterial solution in the sink after the experiment. Place the microtubes and tips in the waste bin on the laboratory bench. Dispose of the bacterial cell suspension liquid and bacterial cell culture waste into a dedicated cell waste container provided in the laboratory. It is desirable to perform the competence procedure aseptically in order to prevent contamination, but in this case, the experiment is performed on each experiment desk up to step 3). In order to increase transformation efficiency, it is important to maintain low temperature at all times during the competence process, so the samples should be stored on ice while not used.
When diluting a solution, make sure to suspend (mix) it before and after dilution, as the cells gradually precipitate.
Apply the undiluted solution to LB medium containing ampicillin, and 105 diluted solution to normal LB medium. The plates are cultured at 37°C. The number of colonies formed on LB plate containing ampicillin is equal to the number of transformed cells. The number of colonies on the normal LB plates is equal to viable count.
Procedure
1) Bacterial Cultivation (Staff)
Pickup single colony of E. coli cells from a LB plate, inoculate it into 10 ml LB broth, and incubate for 16-20 hours at 37°C while shaking. Transfer 1 ml of culture into 100 ml of fresh LB broth in a 500 mL flask and incubate for 4 hours at 30°C while shaking. Collect 5 ml of culture to disposable, ice-cold polypropylene tube. Cool the culture on ice for over 20 minutes, Harvest the cells by centrifugation at 7,000 rpm for 5 minutes at 4°C.
2) Washing and Resuspending the Bacteria
Prepare an icebox and put the tube containing centrifuged culture inside. Carefully decant the media from the cell pellets. Suspend each pellet in 2 ml of ice-cold 0.1 M CaCl2 and store on ice until centrifugation. Recover the cells again by centrifugation at 7,000 rpm for 3 minutes at 4°C. Decant the fluid from the cell pellets. Add 0.3 ml of ice-cold 0.1 M CaCl2 and resuspend the cell pellets. Collect 100µL of suspended cells into a 1.5 ml tube and store on ice.
3) Starting the Transformation
Add 5µL of plUC19（10ng/µL）and mix gently by pipetting。Store the tube on ice for 15 minutes.
4) Heatshock, Heat removal, Addition of LB
Incubate the culture at 42°C for 30 seconds (exactly) in a water bath. Do not shake the tube during incubation。Rapidly transfer the tube to an icebox and chill for 3 minutes。Add 900µL of LB medium to the tube. Incubate the culture at 37°C for 15 minutes in water bath in order to enable the bacteria to express the ampicillin resistance gene encoded by PUC19.
5) Dilution of the transformant, Application, Cultivation
To dilute the cells（105 times）, prepare five tubes with 900µL of 0.9％ NaCl。Dilute the transformant cells。Drop 100µL of x1 (undiluted) cells onto LB medium agar plates containing 100 mg/L ampicillin and gently spread the cells。Drop 100µL of x10 diluted cells onto a normal LB agar plate and gently spread the cells. Invert the plate and incubate at 37°C.
6) Transplantation (next day)
Working inside of a clean bench, inoculate the colony from the obtained LB plate into a liquid medium. To do this, collect some bacteria on a sterilized toothpick and place the toothpick inside of a test tube containing 2 mL of LB liquid medium with ampicillin (2 test tubes per person). Cultivate overnight at 37°C. After cultivation, store the bacterial liquid at 4°C. The samples will be used for the plasmid isolation experiment.
7) Colony counting
Count the number of colonies obtained on the LB plate containing ampicillin (undiluted solution) and LB only plate (105 diluted solution). Calculate the transformation rate and the percentage of bacteria that underwent transformation.
Results
The number of colonies that grew on the LB medium plate containing ampicillin, where the undiluted (x1) solution has been applied, was 383.
The number of colonies that grew on the LB medium plate, where the 105 times diluted solution has been applied, was 159.
Transformation Rate Calculation
The number of bacteria that underwent transformation is represented by CFU-TF, which is equal to the number of colonies on the ampicillin plate (383). Transformation rate is the number of colonies formed when 1µg of plasmid DNA is used. The units are CFU-TF / µg of plasmid DNA. In this experiment, 5µL of plUC19（10ng/µL）, 100µL of suspended cells and 900µL of LB medium were used to make the original solution, and 100µL of that solution was applied to LB plate. Therefore, we can calculate the mass of plasmid DNA applied to a single LB plate (for the undiluted solution).
Percentage of bacteria that underwent transformation
The number of colonies on the LB only plate is equal to the viable count. From the viable count, we can find the value of all cells on which transformation was attempted (CFU-ALL). As the solution was diluted 105 times before being applied to the LB only plate,
CFU-ALL = viable count ×105 = 159×105 .
Using this value, we can calculate the percentage of bacteria that successfully underwent transformation.
Discussion
As we can see from experimental results, only a very small amount of plUC19 plasmid DNA is needed for transformation of many E. coli cells. Using only 1µg of plasmid DNA, as many as 7.7 ×104 colonies of bacteria that is immune to ampicillin can be formed.
However, only a tiny fraction of E. coli undergoes transformation successfully and becomes capable of growing on medium containing the antibiotic that would normally kill it. Based on the experimental results, as little as 2.41 ×10-3 % of E. coli incorporate the plUC19 plasmid DNA in their genetic material correctly.
Perhaps this value is so low because of the insufficient treatment of the bacteria before the introduction of the plasmid DNA. Only the competent cells are capable of incorporating a plasmid, so if the competency process proved ineffective for a part of the population, the values of the transformation rate and the percentage of bacteria that underwent transformation would decrease.
In order to increase the accuracy of the experimental transformation rate value, it would be advisable to increase the number of the investigated samples. Cultivation of E,coli on 5 LB plates containing ampicillin and 5 LB only plates is likely to yield a more reliable result, reducing the impact of random error on the experiment, thus increasing the accuracy. Furthermore, in order to increase the reliability of the results, the experiment should be performed by several independent teams/scientists.
Conclusion
Over the course of this experiment, we have produced competent E. coli cells and succeeded at incorporating plasmid DNA into their genetic material, making the bacteria immune to ampicillin. Based on the experimental results, rate of transformation is equal to 7.7 ×104 CFU-TF/µg of plasmid DNA, but only 2.41 ×10-3 % of E. coli undergoes the transformation successfully. While the influence of random error on the experiment can be considerable and there is a lot of field for improvement, through obtaining the aforementioned two values and successfully inducing transformation in E. coli, the objective of the experiment has been fulfilled.