Pglo Transformation

Introduction
In this week’s laboratory period students had the opportunity to perform a common procedure preformed by many if not all microbiologists known as genetic transformation. Genetic transformation is the ability to move DNA into an organism and thereby altering its genotypic and genetic makeup (2). Genetic transformation has shown to have a wide variety of uses in many scientific studies. In agriculture, gene coding for traits such as frost, pest, or spoilage resistance have been genetically transformed in plants. In the pharmaceutical industry bacteria and yeast are transformed with human genes of interest in order to produce therapeutics for human disorders. An example of pharmaceutical industries use of transformation is seen in the development of insulin, which is used to treat some forms of diabetes mellitus (2). In nature, many strands of bacteria genetically exchange genetic information during a process known as conjugation, and the new information is passed on subsequent generations. The advantage of using bacteria relates to the single-celled nature of the organisms. Only one cell needs to be changed in order to integrate the new genetic information and allow for its transmission to the next generation. The exchange process of genetic information allows the organism to increase in its capacity to adapt to its environment.(2) Genetic transformation is also seen in organisms that are multicellular. However, the process is more difficult and in some cases can be particularly challenging. The multicellular nature of most plants introduces the complication of transforming each cell of the plant in order to fully integrate the new information (2). Often the approach taken in higher organisms, such as plants, involves transforming an individual plant cell and then regenerating it into a whole organism (2). Genetic transformation is not only naturally occurring in plants, but bacteria and viruses are able to carry this out routinely as well. One of these bacteria is Escherichia coli.
E. coli is a strand of bacteria that is commonly found in the lower intestine of warm-blooded animals (3). Most E. coli bacteria strains are harmless, but some are capable of causing food poisoning in humans (3). In addition, the bacteria can also be grown easily and because of its simplicity it can be easily manipulated, thus making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology (3). For this reason, E.coli was the choice of bacteria used for genetic transformation during the laboratory experiment. This process involved the insertion of a new DNA into the E. coli cells. In this case the new DNA was a gene that coded for Green Fluorescent Protein (GFP) which, following the transformation procedure would be expressed in the bacteria causing them to glow a brilliant green under an ultraviolet light. Furthermore, the purpose of this experiment was also to teach students the process of moving genes from one organism to another with the aid of a plasmid. In addition to have a chromosome, bacteria also contain one or more small round pieces of DNA called plasmids (1). Plasmid DNA usually contains genes for more than one trait and through genetic engineering, scientists have been able to insert genes coding for new traits into the plasmid. In lab, students used a plasmid called pGLO which carries the GFP protein which can be switched on in transformed cells by adding sugar arabinose to the cells’ nutrient medium and a gene that allows E.coli to be resistant to ampicillin (1). Cells that have been successfully transformed with pGLO DNA are seen by the amount of cellular growth observed on the antibiotic plates (1). The laboratory experiment required four different LB nutrient agar plates. Each plate contained a different combination of either (+) or (-) pGLO, ampicillin and arabinose. Each plate contained the following: Plate 1 contained LB, ampicillin, and +pGLO, Plate 2 contained LB,ampicillin, arabinose, and +pGLO, Plate 3 contained LB and ampicillin and Plate 4 contained only LB and –pGLO. Based on these environments it was predicted that the plates containing ampicillin would show bacterial growth thereby indicating E. coli’s resistance to ampicillin. In addition, the bacteria grown in plate 2 would also glow a green fluorescence when put under a ultraviolet light, however, plate 1 would not have this characteristic because of the absence of arabinose, the transformation solution. Lastly, it was also predicted that plate 3 would show a lawn of grown while plate 4 would show no growth since it did not provide any nutrients needed for bacterial growth, thus making plates 3 and 4 the control plates.
Materials and Methods
Supplies and Materials
• 1 E. coli starter plate
• 4 poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara)
• 1 Transformation solution
• 1 LB Nutrient Broth
• 7 Inoculation loops
• 5 Pipets
• 1 Foam microtube holder/float
• Container full of crushed ice
• Permanent Marker
• 1 vial of rehydrated pGLO plasmid
• 42°C water bath
• Thermometer
• 37°C incubator

Prior to the beginning of the laboratory session, several needed materials were prepared for students by the teaching assistants and by the professor. These included the preparation of the E. coli starter plate from which students were to obtain their bacterial sample, the transformation solution, LB nutrient broth, and (+) and (-) pGLO solutions. The two samples of pGLO solutions were present in the microtubules and foam rack upon arrival to lab. The first thing that students needed to do was to obtain all necessary materials and label everything properly with their names/initials, dates, and laboratory period. This was done in order to avoid any type of error and confusion from occurring during the experiment. Next, one of the pGLO tubes was obtained and using a sterile pipet, 250 μl of transformation solution (CaCl2). When completing this part of the procedure the students were told to transfer the solution into one pGLO tube ( (+) and (-) ) at a time and also to use new pipets for each transfer. This was done in order to eliminate contamination of the products. These tubes were then placed into the ice tube. From the E. coli starting plate, a single colony of bacteria was obtained using a sterile look. This bacterium was immersed into the bottom of the tube containing +pGLO. Spinning the loop enabled the colony to be dispersed in the transformation solution. This procedure was repeated for the tube containing (-) pGLO. After these samples were prepared both pGLO DNA solutions were examined under a fluorescence light and any changes in color were recorded. A sterile loop was then obtained and immersed into the pGLO plasmid DNA stock tube. Making sure there was a film of plasmid solution across the ring, the sample of pGLO plasmid DNA was put into the +pGLO tube. Students were instructed not to add plasmid DNA to the (-) pGLO tube. These two samples were put into the ice bath making sure that they were all the way down in the rack, and incubated for 10 mintues. After 10 minutes, both (+) and (-) pGLO tubes were put into a water bath that was 42°C in temperature for exactly 50 seconds. The time period of 50 seconds was strictly enforced by the teaching assistants and by the instructor because if the sample remained any longer than required the bacterial samples would alter the results of the experiment. After 50 seconds both tubes were placed back on the ice and incubated for 2 minutes. The tubes were then removed from the ice and placed on the bench top for further use. Taking one of the tubes and using a new sterile pipet, 250 μl of LB nutrient broth to the tube and reclosing it. This step was repeated for the other tube using a new sterile pipet. Both samples were then incubated for 10 minutes at room temperature and mixed by finger tapping. Using a new sterile pipet for each tube, 100 μl of transformation fluid and control suspension was added onto the appropriate nutrient agar plates. The following is a representation of the plates and its components:

Students were strongly encouraged to remember to use new sterile pipettes for each transfer. Each of the suspensions was spread evenly around the surface of the LB nutrient agar using a new sterile loop for each plate. Each plate was then appropriately covered, stacked up, taped together and placed upside down into a 37°C incubator for a period of 24 hrs. The next day students were to return and record their observations. The last part of the entire procedure was to calculate the transformation efficiency through a series of calculations obtained from results from plate #2.
Results
Each of the plates was collected from incubation and the following observations were made for the plates.
Plate #1: +pGLO, LB/amp This plate contained a positive form of pGLO plasmid, as well as the nutrient broth LB and the antibiotic ampicillin. After 24 hrs of incubation, growth of cellular colonies was observed. These colonies were muggy white in color just as the colonies that developed in plate #2, however when tested for green fluorescence under an ultraviolet light they tested to be negative. Also, a total of 93 bacterial colonies were recorded.

Plate #2: +pGLO, LB/amp/ara This plate also contained a positive form of the pGLO plasmid, nutrient broth LB, the antibiotic ampicillin, and arabinose. Comparing plates 1 and 2, the second plate showed more bacterial growth and after counting the actual number of colonies, 119 colonies were observed. The colonies were the same in color as those in plate 1, a muggy white. Also, when the agar plate was flashed under and an ultraviolet light the colonies shined a green light.

Plate #3: -pGLO, LB/amp This plate contained a negative sample of pGLO along with LB nutrient broth and the antibiotic ampicillin. No colonies were present on the agar plate, however a lawn of bacteria was present instead. The bacterial lawn showed to be muggy white in color.

Plate #4: -pGLO, LB This plate contained –PGLO and nutrient broth LB, and so no bacterial growth was observed. The plate came out to be empty with no growth present.

Transformation Efficiency In calculating the transformation efficiency of the results obtained, the number of colonies grown on plate #2 needed to be counted. The plate #2 showed a growth total of 119 colonies. Next the total amount of gGLO plasmid DNA was determined by using the following formula:
(DNA in μg) = (concentration of DNA in μg/μl) x (volume of DNA in μl)
A total of 10 μl of pGLO at a concentration of 0.08 μg/μl was used, and so using the formula above a total of 0.08μg of pGLO DNA was used. Next the fraction of pGLO plasmid DNA (in the bacteria) that actually spread onto plate #2 was determined from dividing the volume spread on LB/amp plate (μl) by the total sample volume in the test tube (μl). From the procedure, it is known that 100μl of cells containing DNA were spread from a test tube containing 510μl of solution. Using the above information the caculation representing the fraction of DNA was as follows:
Fraction of DNA used = 100 μl DNA celss / 510 μl total sample
= 0.1961
Using this number as well as the total amount of pGLO DNA, the amount of micrograms of pGLO DNA spread (μg) was determined by the following formula: pGLO DNA spread in μg = Total amount of DNA used (μg) x Fraction of DNA used
(0.80 μg)(.1961) = 0.15688 μg
Now that all necessary data was collected, the transformation efficiency was calculated by taking the total number of cells growing on the agar plate and dividing that by the amount of DNA spread on the agar plate. This formula was solved in the following manner:
Transformation Efficiency = Total # of cells growing on agar plate/Amt. of DNA spread
= 119 / 0.15688 = 7.58 x 102

Discussion Prior to beginning the experiment predictions pertaining to colony formation were made. It was predicted that the plates containing ampicillin would show bacterial growth thereby indicating E. coli’s resistance to ampicillin. In addition, the bacteria grown in plate 2 would also glow a green fluorescence when put under a ultraviolet light, however, plate 1 would not have this characteristic because of the absence of arabinose, the transformation solution. Lastly, it was also predicted that plate 3 would show a lawn of grown while plate 4 would show no growth since it did not provide any nutrients needed for bacterial growth, thus making plates 3 and 4 the control plates. Based upon the collected results, the predictions came out to be correct. The most significant results were seen in plates 1 and 2, in which both mediums contained the +pGLO plasmid, LB nutrient broth and the antibiotic ampicillin, however, the only difference is that plate 2 contained the transformation solution arabinose. While observing both plates, several characteristics of E. coli remained unaltered. These characteristics included the white color that the colonies appeared to be, the size of the bacterial colonies, and their overall morphological characteristics remained unaltered, such that if the bacteria were to be tested using gram stain they would consistently show the same results. Furthermore, from the results, two new traits were observed and those included the bacteria’s resistance to the antibiotic ampicillin and the ability of the bacteria to glow in the presence of an ultraviolet light (Plate 2). The pGLO plasmid used in both plates encodes the gene for GFP and the gene for resistance to the antibiotic ampicillin, therefore, when both plates 1 and 2 were exposed to these materials bacterium grew signifying its resistance to ampicillin. This showed that when genetically transformed cells acquire the ability to live in the presences of the antibiotic ampicillin, then the other genes present on the plasmid must have also entered the transformed cells allowing them to acquire the same characteristic. Such physiological abilities shown by the bacteria gives them advantages, because by being able to switch particular genes on and off enables them to better camouflage themselves increasing their likelihood of survival and it also allows the organism to know what is available and beneficial in the environment, and how to use the nutrients given. Despite these similarities differences between the two plates were also observed such that only plate 2 containing the sugar arabinose allowed GFP to be switched on during transformation permitting cells to glow a light green color in the presence of an ultraviolet light. The glowing of the bacteria indicated that the cells transformed during replication due to the presence of the sugar arabinose. Therefore, without arabinose the bacterial would not be able to glow fluorescent green, and so when the UV light was shined onto the sample of plate 1 containing the original pGLO plasmid DNA no fluorescence occurred. This indicates that in plate 1 only the protein GFP was present and this sample was lacking the plasmid DNA and DNA polymerase needed for glowing to occur. Also characteristics presented in the wavelengths of the UV light were able to absorb those of arabinose allowing the green color to be shown. With these observations the plasmid DNA and the transformation solution were eliminated as being a source of fluorescence and furthermore, confirming the protein GFP as being the source of fluorescence. An additional set of plates was prepared using –pGLO plasmid. Plate 3 contained –pGLO plasmid, LB nutrient broth, and ampicillin, while plate 4 contained –pGLO and LB nutrient broth only (Figures 3 and 4). These two plates were used as control plates, such that comparisons of results collected from plates 1 and 2 were compared to those obtained from plates 3 and 4. Plate 3 showed growth in the form of a lawn that was muggy white in color. This signified the presence of E. coli whose population spread throughout the plate, and its use of ampicillin as a growth nutrient. Furthermore, it signified E. coli’s resistance to the antibiotic. Plate 4, contained no source of viable nutrients that could be used for growth therefore, no significant results appears on the agar plate.
The last significant characteristic that was observed was the amount of bacterial growth that occurred within the plates. Plate 1 has a total growth of 93 colonies; plate 2 had a total of 119, while plate 3 showed to be a lawn and plate 4 showed no sign of growth. Furthermore, this quantitative information was essential in calculating transformation efficiency, which determines the effectiveness of transforming DNA molecules into bacterial molecules. Although, both plates 1 and 2 showed bacterial growth (Plate 1=93 colonies, Plate 2=119 colonies), only the number of colonies formed in plate 2 was considered since their results showed the success of transformation. A total of 119 colonies were collected, and after all necessary calculations were completed, a transformation efficient of 7.58 x 102 was obtained. According to the lab handout, biotechnologists agree that the transformation protocol has transformation efficiency between 8.0 x 102 and 7.0 x 102. The experimental value collected fell in this range therefore showing to be significant.
There are several approaches that could be taken in order to distinguish whether a bacterium is truly resistant to an antibiotic, in this case to ampicillin. One of those methods includes changing the environment in which they grow, more specifically by using differential and or selective plates. By using differential media, researchers would be able to identify different strains of bacteria based on their ability to utilize certain nutrients to which they are exposed. By using selective media, researchers would be able to determine the genetic character of a microbe by its ability to grow in the presence of a selective agent or selective conditions. Information collected from either growth methods would benefit researchers in finding more information about the different strains of bacteria, which exist.

References
1. Student Manual:PGLO Transformation. DePaul Univeristy. 2008. 28-48.
2. What is Genetic Transformation? Saskatoon: AG-WEST BIOTECH INC., 2001.
3. Escherichia coli. (2008, May 11). In Wikipedia, The Free Encyclopedia. Retrieved 03:33, May 12, 2008, from http://en.wikipedia.org/w/index.php?title=Escherichia_coli&oldid=211679577

References: 1. Student Manual:PGLO Transformation. DePaul Univeristy. 2008. 28-48. 2. What is Genetic Transformation? Saskatoon: AG-WEST BIOTECH INC., 2001. 3. Escherichia coli. (2008, May 11). In Wikipedia, The Free Encyclopedia. Retrieved 03:33, May 12, 2008, from http://en.wikipedia.org/w/index.php?title=Escherichia_coli&oldid=211679577