Transformation Efficiency

Bacterial Transformation
Transformation of E. Coli with a Green Fluorescent Protein Plasmid efficiency as measure by number of transformed cells per (ug)

Introduction: Transformation is the process of transferring a phenotypic trait from a donor cell to a recipient
cell. The landmark transformation experiment occurred when Fred Griffith transformed a strain of Streptococcus pneumoniae from a rough colony morphology and an avirulent course of infection in mice, into a strain with a smooth colony morphology and virulent course of infection in mice (1). Later, Oswald Avery demonstrated that the transforming agent was DNA (2). Griffith and Avery’s experiments were critical in demonstrating that DNA is the genetic material. We now know that DNA fragments from the donor cell enter the recipient cell and become incorporated into the recipient’s genome. Any genes carried by the DNA fragment are available for expression in the recipient. In recent years, the term transformation has been broadened to mean not only the transfer of traits, but also the transfer of DNA itself. A cell that is capable of being transformed is called "competent." A few species of bacteria (including Acinetobacter calcoaceticus) are naturally competent, and many other species can be rendered artificially competent in the laboratory. Exposure to calcium chloride followed by a cold shock is one common procedure to induce competence in bacteria. Grampositive bacteria produce a "competence factor" that induces competence in surrounding cells. Grampositive cells under the influence of competence factor will take up DNA from a wide range of sources. Gramnegative bacteria lack competence factors and, in nature, will take in DNA only from closely related cells. Acinetobacter calcoaceticus is a Gramnegative bacillus commonly found in water and soil and occasionally in diseased animals. Some strains of A. calcoaceticus are resistant to the antibiotic Streptomycin. A single gene on the chromosome of resistant bacteria codes for this resistance.

Hypothesis: Due to the addition of the ampicillian, to the agar medium, the number of transformed cells will be greater in comparison to the control group as measured by the number of transformants per ug.

Dependent Variables: The number of transformants at the end of the experiment.

Independent Variables: The ampicillian that was added to certain dishes.

Procedure/ materials:

First put your initials or group number on the tubes labeled "pGAL DNA" and "Control Buffer". Place them back on ice. To set up the control use a fresh, sterile 1 ml pipet, transfer all the cell suspension (0.3 ml) in the tube "Cells for Control" to the tube "Control Buffer". Carefully place the pipet back into its wrapper and save for next step. Cap the tube; mix by tapping. Put the tube back on ice. To set up the transformation use the same pipet from Step 2, transfer all the cell suspension (0.3 ml) in the tube "Cells for DNA" to the tube "pGAL DNA". Discard pipet. Cap the tube; mix by tapping. Put the tube back on ice. Incubate the cells prepared in the beggining steps on ice for 10 minutes. Place the cells in 42°C water for 90 seconds and with the completion of this task, place the tubes back on ice for 1 minute. The next step is to use a fresh, sterile 1 ml pipet to add 0.75 ml of the recovery broth to the tube "Control Buffer". Use the same pipet to add 0.75 ml of the recovery broth to the tube "pGAL DNA". Incubate the closed tubes in a 37°C waterbath for 30 minutes then remove the tubes from the waterbath and place them on the lab bench. Another crucial step is to label the bottom of the 3 agar plates in the following. Put your initials or group number on all the plates, label the X-GAL plate (unstriped) "Control 1". Label one of the AMP/X-GAL (striped) plates "Control 2" Label the second AMP/X-GAL (striped) plate "DNA". In order to plate cells from the tube labeled "Control Buffer": Use a fresh, sterile 1 ml pipet to transfer recovered cells from the tube "Control Buffer" to the middle of the following plates and add 0.25 ml to the plate labeled "Control 1" (X-GAL) and 0.25 ml to the plate labeled "Control 2" (X-GAL/AMP) Spread the cells with an inoculating loop, then cover both control plates and allow the liquid to be absorbed. To plate cells from the tube labeled "DNA"; use a fresh sterile 1 ml pipet to transfer recovered cells from the tube "pGAL DNA" to the middle of the following plate: 0.25 ml to the plate labeled "DNA" (X-GAL/AMP) spread the cells with an inoculating loop and cover the plate and allow the liquid to be absorbed.To preparing plates for incubation stack your group's set of plates on top of one another and tape them together. Put your initials or group number on the taped set of plates. (The plates should be left in the upright position to allow the cell suspension to be absorbed by the agar.) Lastly Place the set of plates in a safe place designated by your instructor. After the cell suspension is absorbed by the agar for approximatelyone hour, you or your instructor will place the plates in the inverted position (agar side on top) in a 37°C bacterial incubation oven for overnight incubation (15-20 hours). The plates are inverted to prevent condensation on the lid, which could drip onto the culture and interfere with experimental results.

Data Collection and Presentation
(Raw Data)

Number of Transformant

Colonies ___________Transormants

(with amp only)Group 1_________ 15 ug_____________ 30 ug
(with amp only)Group 2_________ 3 ug______________ 6 ug
(with amp only)Group 3_________ 28 ug_____________ 56 ug
(with amp+other)Group 4_________ 71 ug_____________ 142 ug
(with amp only)Group 5_________ 5 ug______________ 10 ug
(with amp only)Group 6_________ 28 ug_____________ 56 ug
(with amp only)Group 7_________ 4 ug______________ 8 ug
(with amp only)Group 8_________ 29 ug_____________ 58 ug

Table 1

• Table 1 is a table showing 8 different test groups with varying results of initial bacteria counts and transformed bacteria counts.
  

Data Transformation

Mean: 22.875 45.75

Standard Deviation: 22.48451848 44.96903697


Qualitative Data: Some qualitative data that we had found was the actual color change in the colonies that had the ampicillian added to them. We also noticed that the agar had a very strong smell once we removed the plated pitri dishes from the incubator. We were not able to give a quantitative measurement to the smell of the E.Coli bacteria but descriptions such as strong and pungent were used when referring to the smell. In addition the agar had turned a deeper yellow color in comparison to the color of the bacteria and agar medium before incubation. Lastly there was a significant amount of condensation on all four of our pitri dishes ranging from translucent to almost opaque.



Conclusions
My hypothesis that due to the addition of the ampicillian to the agar medium, the number of transformed cells will be greater in comparison to the control group as measured by the number of transformants per ug was not supported by the data gained from the experiment. Out of the eight trials done seven of them only saw a bacterial transformation while the ampicillin was present within the agar. There was only one group who had results with plates that were not labeled ashaving ampicillin. The standard deviation for the total amount of trials is 22.4 which is high. This tells me that there was a lot of extremes in this experiment and there would need to be a much smaller number of standard deviation to really support my hypothesis. The variations found in the data are to high and frequent for my hypothesis to be supported.

Uncertainties/Limitation

During this lab many small errors were made. When preparing the broth/agar we should have made sure ALL agar was melted. We still had a large piece of solid agar in the middle of the bottle. For preparing the ingredients we had already given them a frozen bath and we had not yet placed the DNA into the lil capsule. While Mr. Ghosh prepared the slides for us, he wasnt sure if all dishes had the appropiate things inside. Lastly, the simple and common error is measurements.

Modifications

There are a number of modifications that can be made to this experiment to make it more efficient and accurate. To make transitions between steps easier and faster the materials could be lined up in order from first step to last step. This would not only create a faster way to complete the experiment but it will also cut down on the possibilities for human error because the distance for carrying and handling will decrease. Also labeling accurately will also help to keep track of all test trials and variables in order to increase the integrity and validity of end data.