The objective of this transformation experiment was to pick transformed bacterial cells from the resistant cells. The E. coli cells were engineered to take up the pAMP plasmid with the ampicillin resistance gene. Some E. coli cells did not take up the pAMP plasmid. Other E.coli cells were treated with the T. E. buffer and allowed to grow on the plates of agar. The bacterial cells that incorporate the plasmid DNA become resistant to ampicillin.
Cells transformation is a versatile tool that is widely used in genetic engineering. Also, cell transformation is of critical significance in the progress of molecular biology. The purpose of the cell transformation procedure is to introduce foreign plasmids into bacteria, which then amplifies the plasmids making large amounts of the inserted plasmids. A plasmid is a small piece of circular DNA that contains significant genetic information for bacterial growth. Bacteria, which frequently grow in the same conditions as fungi and molds, developed to make proteins that inactivate the toxins that these organisms produce. The bacteria share these essential proteins bypassing the information amongst them as plasmids carrying genes. As a result, the natural role of the plasmids is to transfer the genetic information that is required to the bacteria survival. It is this trait of a plasmid that is exploited in the transformation of bacteria.
For the E. coli to take the plasmid, it should first be made competent to incorporate and express the plasmid DNA. E. coli is an organism that is broadly used in recombinant DNA technology and the entire world of science because of its ability to grow swiftly in most mediums. The experiment is based on the idea of transformation of the E. coli cells enabled to take up and express the plasmids carrying two genes. One of the gene codes for ampicillin-resistance and the second gene codes for a GFP (green fluorescent protein). The experiment presents the theory of recombination and the data obtained from the experiment can have profound effects on the ampicillin resistance in human bodies. The ampicillin resistance gene allows for the selection of the transformed E. coli cells based upon their ability to grow up in an environment containing the antibiotic ampicillin.
The aim of the experiment was to transfer the pAMP gene into the E. coli cells. The hypothesis of the experiment stated that the bacteria would become resistant to ampicillin upon the incorporation of the ampicillin gene. The goal of the experiment was to understand the techniques of the recombinant DNA, particularly, the transformation process. Recombination of genes includes three main principles, namely; conjugation, transduction, and transformation. The transformation process uses the plasmids as vehicles to carry the foreign genes into the E. coli. The plasmid DNA was directly absorbed into the E. coli cells together with the donor DNA contained in it. The pAMP was the “donor cell” in this experiment. The experiment utilized the idea of genetic engineering to show how the recombination of genes takes place naturally.
Materials and Equipment
- Disposable gloves
- Microtube rack
- Two microtubes
- Bacterial waste container
- Sterile plastic tips of the pipette
- Agar plates; LB/Ampicillin, LB plate, LB/Amp/Arabinose
- Two plates with LB positive medium ampicillin
- Two plates with LB medium
- Six disposable inoculating loops
- Six disposable pipettes
- Waterproof maker
- The pAMP plasmid
- TE buffer
- Plates with E. coli cells grown overnight and streaked out
- Water bath
- Crushed ice
- GREEN plasmid (0.005 µg/µl)+
- Distilled water
- Luria broth
- UV light
- Competent cells kept on ice
- 37°C incubator
The first experimental measure is a thawing of the competent E. coli cells on crushed ice. The labeling of the Eppendorf tubes; one with a negative (-) followed and the other with a positive (+) signs and the names of each table written on the tubes. The placement of 50microliters of the competent E. coli cells into each microtube followed the thawing of the competent cells. The next step was the adding of 10microliters of the pAMP plasmid into the tube labeled positive (+) and the addition of 10microliters of the T. E. buffer into the tube labeled negative “-”.
The incubation of the tubes containing the mixture of ice for fifteen minutes followed. While waiting for the incubation to complete, the two LB plates of agar were labeled, one with positive (+) and the other plate with a negative (LB-). The LB Amp (-) and the LB Amp + labeled the other plates of agar. Letters that denoted the students' tables were used to label the Plates of agar. Following the fifteen minutes of incubation, the cells were heat-shocked at 42°C for one and half hour. The returning of the cells into the ice for two minutes followed the heat-shocking.
The next step included the addition of 200microliters of the Luria broth into each tube. In order to have the material mix properly, a finger was used to gently tap against the tubes. The next step was the incubation of the tubes containing the mixture at 37°C for 45 minutes. Following the incubation, 70microliters of “+” cells were placed on the center of the LB (+) plate of agar and a sterilized L shaped glass rod was used to spread the cells on an agar plate. Next was the placement of 70microliters of the (-) cells on the center of the LB (-) plate of agar and the sterile L shaped glass rod was used to spread the cells on an agar plate. The plates were then allowed to sit for five minutes. Lastly, the whole class taped all the plates together and labeled the plates with the name of the TA and incubated the inverted plates at 37°C overnight.
| Positive(+) Cells
Treated with pAMP
|Growth present in the form of colonies
| Negative (-) Cells
Treated with T. E. Buffer
|No growth present
The cells that became transformed using the ampicillin resistance gene were able to stay alive and grow in the medium. There was growth on the three of the four agar plates, and only one plate, the LB/Amp (-) plate, did not contain any growth on it.
The outcomes of this experiment showed that the pAMP gene was capable of successfully recombining with the E. coli cells. Consequently, the colonies made following the recombination of the pAMP plasmid DNA and the E. coli cells were able to flourish on the agar plates containing the ampicillin. The E. coli bacteria grow regularly; however, the presence of the ampicillin antibiotic hindered the growth. The E. coli cells survived and grew in the presence of the ampicillin antibiotic because of the pAMP gene contained in the plasmid, with which the E. coli recombined. The addition of the Luria broth into the plates introduced the E. coli cells into the pAMP gene. The E. coli cells took up the pAMP plasmid containing the foreign gene and the cells become transformed. Because of the taking up of the foreign DNA by the E. coli cells, the end product included the transformed E. coli cells that were resistant to the ampicillin antibiotic. The resistant E. coli cells grew in the medium containing the antibiotic.
The hypothesis of the experiment was proven right with the E. coli transformation. The hypothesis clearly stated that the transfer of the foreign genes into the E. coli cells would result in the ampicillin resistance. Such hypothesis proved right by the results of the experiment, which gave rise to the E. coli cells that were resistant to the ampicillin antibiotic. The results clearly indicated that the E. coli cells were capable of surviving on three of the four plates of agar on the mediums containing the ampicillin antibiotic. The E. coli cells placed on the LB/Amp (-) plate of agar was the only cells that failed to produce. The failure of these cells to grow on the ampicillin containing medium means that the cells did not take up the donor pAMP gene and so they did not transform. As a result, the growth of these un-transformed E. coli cells was hindered by the ampicillin antibiotic, so that they failed to survive and grow on the medium containing the ampicillin. Consequently, there were no colonies of cells.
Although the results of the experiment matched with the hypothesis, there were many chances of errors occurring in the course of the experiment. The timing process was one good source of error. The experiment needed the materials to be timed at certain stages of the procedure and the incubation also required to be done for a specific period. Error or errors in timing could have resulted in any of these timing instances. Another likely source of error could be the process of sterilizing the materials such as the L shaped glass rod. Improper sterilization of the equipment can lead to major contamination effects. Considering the possible mistakes, questions arise as to whether the ignored or not observed errors can be detrimental to the experiment results. However, if there were errors in the experiment, more than gene recombination could possibly occur.
Eventually, the results of the experiment went back to justifying one of the major scientific concepts of recombination, transformation. Such experiments may lead to considerably novel findings in science. The breakthrough of new theories from simply accidental errors in an experiment constitutes a great part of science. The future of science holds many more fresh ideas to discover and more experiments to carry out.