In this lab, the goal was to transform the bacteria e-coli to glow in the dark (or under a black light). Four plates were set up with agar in them for the bacteria to feed on and grow. Changes were then made to the bacteria. One plate was the control plate, having only the LB or agar for the bacteria and negative pGLO, which is the liquid not containing the plasmid. This is the plate that was compared with the three others in order to identify the changes. The second plate contained negative pGLO, LB, and ampicillin. This is to see whether or not the bacteria will become resistant to the ampicillin and grow, or not be resistant and have no growth. The third plate contained positive pGLO (has the plasmid), LB, and ampicillin. Again, to whether there is resistance to the antibiotic with the difference of a positive pGLO rather than the negative pGLO. The fourth plate contains the positive pGLO, LB, ampicillin, and arabinose (a sugar). One of these plates will have the ability to glow in the dark because the gene making GFP (green fluorescent protein) will be turned on.
The whole idea of this gene being turned on comes from the outside of this lab and within the anatomy of the bacteria itself. This bacteria goes through the process of gene regulation during transcription. This process turns on only a fraction of the cells genes. When genes are turned off, it is because a repressor binds to an operator in the cell, which stops the process of transcription. This occurs when high levels of tryptophan bind to the repressor causing it to bind again to the operator. When a gene is turned on, a metabolite binds to the repressor, which stops it from binding to the operator and allows transcription to continue, turning the gene on. In one of these plates, the gene will be either turned on or off based on what is added to the bacteria.
Materials and Methods
See science manual Bacterial Transformation Lab for complete list of materials and procedures.
There are colonies because the pGLO contains the plasmid, which allows the bacteria to survive and become resistant to the ampicillin. Has a beige color
Also contains pGLO with plasmid, so the bacteria is resistant to the ampicillin
Arabinose is also present
The bacteria on this plate glow in the dark almost a greenish color.
No bacteria at all
The plasmid was not present, therefore the bacteria was not resistant to the antibiotic
TNTC, plate is full of beige bacteria.
Based on the results, there are conclusions that can be made about what is seen. The plate with +pGLO LB/Amp was resistant to the antibiotic. There were colonies all over the plate due to the resistance. This comes from the plasmid in the pGLO, which allows the bacteria to transform and become resistant to the ampicillin. The plate with +pGLO LB/Amp/Ara is the plate with the bacteria that glows. As explained in the introduction, this is because the gene to make GFP was turned on. The arabinose put into the bacteria helped to make the GFP.
Without the arabinose, the bacteria would not be able to make the GFP. This plate also expressed resistance to the ampicillin like the first plate. The third plate contained pGLO LB/Amp. This plate showed no bacteria at all. This is because there was no plasmid to make the bacteria become resistant. In the fourth plate, the bacteria count was TNTC because it only contained pGLO and LB, there was no change made to the bacteria. Analyzes these changes came easy because there was a control plate (-pGLO LB) to compare all the transformations to.
For this lab, there is also many questions and math involved. One question is, which organism is better suited for total genetic transformation- single celled or multi-celled? The best answer is a single-celled organism because that one cell would be able to take up a new gene. Another question is, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish or mouse? The best answer for this question would be a bacterium, since they are single-celled and they reproduce rapidly. There is also a lot of math involved. Starting with the LB/Amp/Ara plate, the amount of cells is 53 found on the plate. To then find the total amount of pGLO DNA (ug), the concentration of DNA (ug/ul) needs to be multiplied by the volume of DNA (ul).
In this case .08 x 510, summing up to 40.8 ug. The next step is to find the fraction of DNA used by dividing volume spread on LB/Amp plate (ul) by the total volume in the test tube (ul). This would be 100 / 510, summing up to 10/51 or .2. After, the pGLO DNA spread (ug) must be found by multiplying the total amount of DNA used in ug by the fraction of DNA used. So, 40.8 x 10/51, this sums to 8.16 ug. This leads to our ultimate goal of the transformation efficiency by dividing the total number of cells growing on the agar plate by the amount of DNA spread on the agar plate. This would be 53 / 8.16, summing to 6.5 as our transformation efficiency.
Overall, the lab was very successful. All the results came out as they were supposed to. The +pGLO with LB/Amp/Ara has glowing bacteria due to the transformation it went through. The arabinose helped turn on a gene with transcription in order to make GFP (green fluorescent protein), which was expressed when the UV light was shown onto the bacteria giving it a green glow rather than all the other bacteria. There was a resistance to ampicillin in this plate. In the plate containing +pGLO LB/Amp, there was also a resistance to the ampicillin. This is because a plasmid was present to transform the bacteria into being resistant to the antibiotic present.
In the plate containing pGLO LB/Amp, there was no bacteria found on the plate. This is because a plasmid was not present to transform the bacteria to become resistant. On the plate containing pGLO LB, there was TNTC with bacteria. This is because it is the control plate and nothing in the bacteria or the plate was changed or transformed. From this lab, there were no errors. Everything ran perfectly and smoothly. If this lab was done over again, it would most likely be done on a larger scale. Example, maybe more bacteria growth or more transformations so the changes become really clear. Also, it could be used to see what other genes can be turned on or off. All in all, the lab was fun and a success in transforming the bacteria, e-coli, into one that is antibiotic resistant and into one that can glow.
1)Bacterial Transformation Lab. Parafilm. American National Can Co. 2015.
2)Operons. AP Biology Homework Handouts. Notes. 2015.
3)LabBench. LabBench. Pearson Inc, n.d. Web. 03 Mar. 2015.