The purpose of the experiment was to genetically transform E.coli to make it antibiotic resistant and glow in the dark through genetic transformation. We were also trying to make specific E. Coli glow under a blacklight.
Genetic transformations are when a gene is inserted into an organism to change that organism’s traits. Single celled organisms are better suited for transformation than multicellular organisms because of how quickly they are able to reproduce. Because bacteria is a single cell, it is able to reproduce quicker. Organisms that reproduce quickly are also better suited for transformations because you can see the results of the offspring's traits quicker. E. coli is well suited to genetically transform. E. coli becomes antibiotic resistant when you add the plasmid pGLO to it. The pGLO plasmids encodes the gene for gfp. Gfp is the gene that is resist to the antibiotic ampicillin. PGLO also has a gene regulation system that controls the expression of the fluorescent protein. The gene is switched in when it is the presence of the sugar arabinose. Transformed cells will appear white in normal lighting and fluorescent green in uv lighting.
Method
- First we labeled one test tube -pGLO and another one +pGLO
- Using a sterile pipet we put 250 ul of CaCl2 into each tube and put the tubes on ice
- Then using a sterile loop we picked up an E. coli colony and put the loop into the +pGLO
tube and spun the loop until all the E. coli was off of it. We repeated this with the -pGLO.
- We then used a sterile loop to transfer pGLO plasmid from the stock tube into the +pGLO tube.
- We out the two tubes on ice for ten minutes, and then into a hot water bath for fifty seconds and then immediately back into the ice for two minutes.
- Using a sterile pipet we transferred 250 ul of nutrient broth to big test tubes.
- Using a sterile pipet we transferred 100 ul of -pGLO solution onto a transformation plate with lb, and transferred another 100 ul of -pGLO solution onto a plate with lb and ampicillin
- Using a sterile pipet we transferred 100 ul of +pGLO solution onto a transformation plate with lb and ampicillin, and transfer 100 ul of +pGLO solution onto a transformation plate with lb, ampicillin, and arabinose.
- Using a sterile loop for each plate spread around the E. coli on the plate.
You would expect to find the most growth on the plate labeled lb,amp,ara since the E. coli is antibiotic resistance in that plate and it should have an affinity for the sugar arabinose. The amp and ara are what allows the E. coli to grow as well as glow in the dark. Any genetically transformed E. coli would be the two plates with the +pGLO since the pGLO was added. This should cause the E. coli to be be antibiotic resistant. The plate containing the sugar should have E. coli that glows in the dark. This was the only plate that had that added. We compared the genetically transformed plates to the two plates without pGLO. E. coli should grow on the plate with no pGLO and no ampicillin to show that the E. Coli can grow on their own. There should be no growth on the plate with no pGLO and ampicillin since the ampicillin should kill the E. coli if it's not antibiotic resistant. In the problem above you can see we calculated transmittance percentage. The percentage shows how well the cells were able to express the gene. As shown, just over one half of the cells expressed the genes which is why the E. Coli was not glowing as much because we had such few cells.
In our results, we were able to see a very small amount of E. Coli glowing under the blacklight. This could be due to a lack of sugar within the dish that we placed the E. coli in. However, we were able to see a very small amount We had successful growth in our dishes, so there was most likely no contamination of pipettes that were not sterile. If this were to happen, there would have been growth in E coli that there should not have been. The +pGLO would have mixed with the -pGLO, which could have changed our results. Since this did not occur, we saw growth and had antibiotic resistance in the correct dishes.
In conclusion, we were able to use pGLO on E. Coli in order to make it antibiotic resistant using genetic transformation.The plasmid was able to allow us to move the DNA of the E. Coli interchangeably with the gene that codes for the Green Fluorescent Protein. E Coli was the best organism to genetically transform because it is a single celled bacteria. This allowed it to reproduce more quickly than a multicellular organism, therefore we were able to tell which coli had the most growth in the dishes. We were able to also see the efficiency at which the E. Coli expressed the genes by the transformation efficiency.
