In this lab experiment, E. coli bacteria was being transformed with a gene coding for green fluorescent protein, or GFP, in efforts to make the bacteria glow. The method used to transform the E. coli bacteria with the GFP gene was heat shock, the vector used was the pGLO plasmid, containing the GFP gene and a gene for resistance to the antibiotic ampicillin and the competent cell that was transformed was E. coli. In order for the GFP gene to glow the sugar arabinose, is required to turn on the biosynthesis pathway. It was hypothesized that if E. Coli bacterial cells are transformed with a gene coding for the green fluorescent protein then the E. Coli cells will show the trait. The predictions for the results of this lab, was that the only plate that would glow would be the LB/AMP/ARA plate with pGLO. The significance of the experiment is determining if genetic transformation was done correctly, because genetic transformation is a widely used technique in the field of genetics and medicine.
There are two vectors that are used most often in genetic transformation, E. coli plasmid vectors and bacteriophage ?, with E. coli being the most common used in recombinant DNA technology. Recombinant DNA is any molecule of DNA that is formed by joining DNA fragments from outside sources, by cutting DNA molecules then joining the resulting fragments. Plasmids are double -stranded and circular DNA molecules, meaning that they only work with prokaryotes such as bacteria and yeast, and very few higher eukaryotic cells that also have the circular DNA shape (Lodish et al. 2010).
The GFP gene has been around for over 160 million years, and is found in one species of jelly fish, Aequorea Victoria. The first time it had been cloned was in 1994, and since then GFP has been used in millions on laboratories around the world with species such as flatworms, algae, bacteria and pigs, to make them flow with the gene. Credit for the discovery of the gene was given to Osamu Shimomura, Marty Chalfie and Roger Tsien, along with the Chemistry Nobel Prize in 2008. GFP is such a useful gene because it makes results of experiments obvious, because when the green fluorescent light glows, it is clear if gene transformation in an experiment was successful or not if there is no glow (Zimmer 2017).
Materials and Method:
All procedures from this lab experiment were obtained from Weedman (2016).
A 1000 microliter pipette was used throughout the lab to micropipette various amounts of solutions. Two microfuge tubes were used for the +pGLO and -pGLO solutions. A total of 250 microliters of transformation solution was used, half in each tube. A total of 6 sterile loops were used to collect colonies of E. coli (2) and to spread solutions on the agar plates (4). 1 LB plate, 2 LB/amp plates and 1 LB/amp/ara plate were used to put the -/+ pGLO solutions on. A total of 250 microliters of LB nutrient broth was added to the – and + pGLO tubes, 250 each. 100 microliters of -/+ pGLO was added to each agar plate. Many new tips were used throughout the experiment.
The first steps in the experimental procedure were to collect two microfuge tubes and label them, with +pGLO and -pGLO, put gloves on and gather a 1000 microliter micropipette and a box of fresh tips, then transfer 250 microliters of the transformation solution into both tubes, close the lids and place the tubs on ice. Next was to pick up a single colony of bacteria using a sterile loop and immerse the loop in one of the two tubes, spinning the loop to get the colony off, then repeating with the other tube. Following that was to incubate the two tubes on ice for 10 minutes, while also obtaining 1 LB plate, 2 LB/amp plates and 1 LB/amp/ara plate. The plates were labeled on the bottom and with -pGLO on the LB and LB/amp plate and +pGLO on the other LB/amp plate and the LB/amp/ara plate. After the incubation period, the tubes were placed directly into the floating racks in a 42 C water bath for 50 seconds, after carrying the tubes to the water bath while in the ice beaker. This heat shock increased uptake of plasmid into E. coli by forcing the bacterial plasma membranes to be more permeable to the plasmid solution which enhanced the rate of successful transformation. After the 50 second hot bath, both tubes were immediately placed back on ice for 2 minutes, then placed back on the rack at the bench. 250 microliters of LB broth was added to the +pGLO tube, with a fresh tip, then the same to the -pGLO tube, with another new tip, then both tubes were incubated at room temperature for 10 minutes. After room temperature incubation both tubes were gently flicked to mix the contents. Using a fresh tip each time, 100 microliters of the +pGLO and the -pGLO suspensions were pipetted on to the correctly labeled LB agar plates. The suspensions were spread around evenly and gently on each plate, using a new sterile loop for each plate. Last steps were to stack the plates upside down, tape them and label them.
In this experiment, the bacteria E. Coli was transformed with the green fluorescent protein gene using the pGLO plasmid and arabinose sugar to activate the green glow. The hypothesis was that the LB plate with no pGLO plasmid and the LB/AMP plate and with pGLO plasmid would grow but nothing would glow, the LB/AMP plate without pGLO would not grow or glow and the LB/AMP/ARA with pGLO would both grow and glow.
The results were fairly accurate with what was hypothesized at the beginning of the experiment. The LB/AMP plate with the pGLO plasmid was hypothesized to grow but not glow, but it was observed that there was no growth and no glow. The LB/AMP/ARA plate with pGLO plasmid did glow green and also grew. The LB/AMP without pGLO plasmid did grow and did not glow as did the LB plate with no pGLO plasmids did not grow nor glow.
Plate Hypothesis Observation Agar surface covered
(-pGLO) LB Grow: yes
Glow: no Grow: yes
Glow: no 95%
LB/AMP Grow: no
Glow: no Grow: no
Glow: no 0%
(+pGLO) LB/AMP Grow: yes
Glow: no Grow: no
Glow: no 0%
LB/AMP/ARA Grow: yes
Glow: yes Grow: yes
Glow: yes 10%
Results from in class experiment Hypothesized results
(Black and Wise 2018)
The predictions were that the LB plate with no pGLO plasmid and the LB/AMP plate and with pGLO plasmid would grow but nothing would glow, the LB/AMP plate without pGLO would not grow or glow and the LB/AMP/ARA with pGLO would both grow and glow. It was hypothesized that if E. Coli bacterial cells are transformed with a gene coding for the green fluorescent protein then the E. Coli cells will show the trait.
The results of the lab did support the hypothesis, because the plate with LB/amp/ara and the pGLO plasmid exhibited bacterial cells that were glowing under a UV light. There were about 18 glowing colonies counted on the agar plate with pGLO, LB, amp and ara. The one prediction that did not turn out correctly was that the LB/AMP plate with the pGLO plasmid should have had some bacterial growth, but none was observed after the experiment. On the LB plate with no pGLO there was significant bacterial growth, with about 95% of the plate covered in colonies. There was 0% coverage on both LB/AMP plates, with and without the pGLO. There was about 10% coverage on the plate with the glowing colonies.
The LB/AMP +pGLO and LB/AMP/ARA +pGLO both should have had bacterial growth because the pGLO plasmid contains a gene that is resistant to ampicillin, meaning that the antibacterial will not affect the growth of the bacteria. The LB -pGLO should have had bacterial growth because there was no ampicillin present to inhibit growth, there was only the broth, which serves as a type of food for the bacteria. The LB/AMP/ARA plate with pGLO should have been the only plate glowing because the plate contains the arabinose sugar required for the green fluorescent protein pathway to be activated to allow the cells to grow. The plate containing the pGLO protein on the agar with only LB and AMP did not glow, as there was no arabinose present.
The plate with the pGLO plasmid and LB/AMP was supposed to show some bacterial growth, but none occurred. The reason for this was likely an error in the experimental procedure, whether it was contamination of a pipette tip or loop, or something bigger like placing no pGLO plasmid on the agar instead of placing the plasmid on the plate.
As with any lab experiment among any age of scientists, there is the possibility of error everywhere. Contamination was likely a huge factor in the chance of error in this particular experiment, as any of the solutions, plates, samples, etc. could have been contaminated at any point, which would alter final results and observations. Some weaknesses in the experimental setup and data collection procedures could have been things like miscommunication between the four group members at any point in the process, errors in keeping track of time during the many waiting periods during the process, or forgetting to keep samples on ice while transporting them to and from the lab bench to the heat bath, or at any other time they should have been on ice.
Genetic transformation was successful in this experiment. The hypothesis was entirely correct, and the predictions made for this experiment were right for the most part, meaning that if E. Coli bacterial cells are transformed with a gene coding for the green fluorescent protein then the E. Coli cells will show the trait, in this case the cells did glow. Growth was seen on the LB plate without pGLO plasmids and LB/AMP/ARA with pGLO because the LB serves as an energy source for the bacteria and the pGLO plasmids are resistant to ampicillin. The LB/AMP plate with pGLO should have showed some bacterial growth, but that was an example of where human error played a part and altered results.
Genetic transformation has already proven to be a widely used technique in the field of genetics and medicine, it will continue to expand and allow scientists to make discoveries that will benefit our world.