Introduction
Genetic Transformation is crucial when attempting to see how an organism reacts to new and foreign pieces of genes and DNA. It can be used in biotechnology for gene coding, transforming bacteria, and seeing what diseases are caused by defective genes and how they are treated. In this experiment, the goal is to transform bacteria (E. coli) with genes that link to GFP (green fluorescent protein).
This GFP is what gives off a bioluminescent color that is often found in jellyfish and causes things to glow in the dark. It consists of three amino acids that go in this specific sequence: serine-tyrosine-glycine. This chain folds and causes glycine and serine to have a chemical bond, and over an hour later, oxygen around the chain attacks tyrosine bonds, creating a double bond that makes this bioluminescence present (Goodsell 2003). In order for E. coli to glow, we must turn on GFP with sugar arabinose and a special plasmid called pGlo. This plasmid contains an origin and the gene needed for GFP. Additionally, it holds the gene for arabinose C protein, which also helps this experiment greatly (Blaber 2004).
Plasmids are crucial to bacteria because they help them deal and survive with stress. Plasmids are like “delivery vehicles” for foreign DNA within bacteria and helps scientists modify them for experiments. For scientists they are great DNA delivery vectors because they can easily be forced in bacteria, can be independently copied, and are circular—which helps with DNA sequences (Sci 2014). There are several ways for genes to be transformed and in this experiment, that will be through heat shock.
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This is where cells are placed in a sudden increase of temperature, which ultimately increases plasma membrane permeability and causes a cell to take DNA surrounding it. This will help deliver a specific vector containing the plasmids that, when transforming E. coli and GFP, are needed to create that bioluminescent glow. Therefore, our hypothesis is that if the plate contains all the necessary materials listed above, then it will show the most bacterial colonies (Weedman 2016).
Materials and Methods
For the procedure, we will follow the instructions given in the Life 102 Lab Manual by Weedman (2016). Before mentioning the steps, you should know that you must wear gloves during this experiment, as we are handling bacteria and you would not want that on your skin or in your body. Firstly, with two initialed microcentrifuge tubes (one labeled +pGLO and the other -pGLO), use a micropipette to transfer 250 μl (microliters) of transformation solution into each tube. With the lids closed, place these tubes in ice. With a sterile loop, pick up a single colony of bacteria from a starter plate and put the transformed loop in the bottom of the +pGLO tube.
Spin the loop in the liquid until the colony is dispersed and no chunks are present, and then put it back in the ice bath. Do this same thing with the -pGLO, but with a new sterile loop so you do not mix the contents. With a micropipette, add 5 μl pGLO to the tube labeled +pGLO, NOT the tube labeled -pGLO. Make sure the lids on both the tubes are closed and then let both tubes rest in ice for 10 minutes. During this time, you can take 4 LB plates from your TA and label them according to the specific plates on the bottom: (+pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB).
After the ten minutes is up, place them in floating racks in a 42 degrees Celsius water bath for 50 seconds and then IMMEDIATELY put them back into ice for 2 minutes. After, place tubes on to a rack and with a fresh tip on your micropipette, add 250 μl of LB nutrient broth to +pGLO tube. With another fresh tip, do the same with the -pGLO tube. Close lids and incubate for 10 minutes in room temperature. After 10 minutes, flick the tubes with your finger and with a fresh tip for each tube, micropipette 100 μl on to the plates accordingly in the corner of the plate. (+pGLO on +pGLO plates and -pGLO on -pGLO plates).
Using new sterile loops for each plate, spread the suspensions evenly on the plate with a ‘skating’ motion. Do not press roughly into the agar (the yellowish gel on the bottom of the plate). Stack plates and tape them together and then write name and lab section number. Upside down, the plates should then be places in a 37 degrees Celsius incubator for 24 hours. To determine the results of the experiment, observe the bioluminescence under normal room lighting, and under a UV light in a room with the lights off. Draw your observations.
Results
In this experiment the goal was to transform the E. coli bacteria with genes linked to GFP and pGLO through heat shock in order to create a bioluminescent glow to show if bacterial colonies can grow. With four different plates (+pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB) we allowed the bacteria to grow for a week before we reviewed our results. Only one plate glowed which was the +pGLO LB/amp/ara because all the necessary products are there for it to show bioluminescence. In the +pGLO LB/amp plate, we discovered four glowing colonies and in the +pGLO LB/amp/ara there were 9 that did not glow. In the -pGLO LB/amp there were no colonies found and in the -pGLO LB there was a constant smear over the plate that showed large amounts of bacterial colonies. So, in short, only the -pGLO LB/amp showed no signs of bacterial colonies.
Glow Grow Colonies
- +pGLO LB/amp/ara Yes Yes 9
- +pGLO LB/amp No Yes 4
- -pGLO LB/amp No No None
- -pGLO LB No Yes Many
Discussion
Our hypothesis for this experiment was that the plate containing all the materials needed for bioluminescence—GFP, pGLO, ampicillin, and arabinose—would result in the most growth and glow, showing the largest production of bacterial colonies. This hypothesis was supported in the results, because the +pGLO LB/amp/ara (which contained all the necessary materials) showed the most bioluminescence and bacterial colonies. This is clearly shown in Figure 1, where we can see a total of nine glowing colonies. In Figure 2, we can see that the plate with all these materials was the only one to show bioluminescence, but two other plates also showed growth. The +pGLO LB/amp and the -pGLO LB/amp showed growth but no bioluminescence, which means that for the E. coli bacteria to glow, arabinose is necessary. The only plate that showed no growth or glow was the -pGLO LB because it does not contain any of the materials needed, which supports our hypothesis.
In similar test conducted by Richard Stone to determine bacterial transformation in E. coli, he also uses the same materials we used in this experiment. He uses the pGLO plasmid that contains the needed ampicillin which is used to kill the bacteria. He then uses GFP which when isolated can be “glued” into a plasmid with the ampicillin gene, that later creates pGLO. Just like in our experiment, Stone uses the heat shock method to create a transformation solution. Then there must be the presence of arabinose in order to create the ampicillin resistance in order for the bacteria to survive. This mixture is then put on to different culture dishes, quite like how we performed our experiment. Stone’s results were very similar to our own.
The plate with only LB and no pGLO shows the most growth of bacteria but has no bioluminescence. The plate with no pGLO and only ampicillin and bacteria did not show any growth because the bacteria could not survive. The LB plate with pGLO and ampicillin showed growth but not a significant one because the ampicillin killed off some of the bacteria. Stone uses a different LB plate, however, which contains no ampicillin, but does have bacteria and pGLO, along with what is needed for GFP. This shows growth and bioluminescence like out +pGLO LB/amp/ara plate. Stone uses some different methods while doing this experiment, but regardless our results are very similar (Stone 2018).
There were some problems and weaknesses that arose from this experiment. One of which was the possible contamination of the bacteria. It is possible that when transferring the transformation solution to the different plates, the tip of the micropipette was contaminated, and it mixed the different solutions. It is also possible that when scraping the E. coli, the student did not do it enough and did not evenly distribute the bacteria, which would not show any bioluminescence or growth. There is also the chance that there was not enough blacklight to see the bacteria glow, so the results were not properly recorded.
Overall, we learned that in order for bacteria to grow and show bioluminescence, E. coli must have the pGLO plasmid, arabinose, ampicillin, and green fluorescent protein. The best results were shown in the only plate containing all of these and the plate that showed no results was the one that contained none of these.
