Lab:
Transformation Of E.coli Cells With A Green Fluorescent Protein Plasmid
By: Emily Burdick and Khayla Moore
Introduction:
Bacterial transformation is of central importance in molecular biology. Bioluminescent is obsserved everyday and these observations pales to the light produced by the bioluminescent which is the natural host of the green fluorescent protein, referred to as GFP. A bright burst of light is observed when energy is transferred to the GFP located specialized on photogenic cells. These proteins do not require substrates, other gene products, or co factors. When exposed to a long wave U.V. light, they emit a bright green light that is visible in bacteria transformed by plasmids that contain the gene coding GFP.
Research Question: Done by Khayla
Hypothesis: Done by Khayla
Variables: Done by Khayla
Methods/Materials: Done by Khayla
Data Collection:
Quantitative Data:
Raw Data: This chart is all the groups colonies and transformation numbers as well as the standard deviation and the averages.
Table 1
Transformed Data: This graph shows the number of colonies found on each groups E.coli (purple) and the number of transformations (orange) on each groups E.coli.
Graph 1
Analysis of Data: The number of transformations was greater than colonies but many groups had high error bars as well. There was a direct correlation between the number of colonies and the number of transformations.
Qualitative Data:
It should be recorded that the bacteria could be labeled as a lime green color and that there were some that were joined together. Here is a picture to better describe the e.coli:
Questions & Answers for Guide:
1. Exogenous DNA does not passively enter E.coli cells that are not competent. What treatment do cells require to be competent?
- A recovery broth is required for cells to be competent.
2. Why did the recovery broth used in this experiment not contain ampicillin?
- The recovery broth used in this experiment did not contain ampicillin because it would have contaminated the control plates.
3. What evidence do you have that transformations was successful?
- The evidence that proves that transformations were successful was that the florescent color that appeared in the e.coli plate.
4. What are some reasons why transformations may not be successful?
- Some reasons why transformation may not be successful are cross-contamination between the plates, the DNA did not effect the e.coli, or simply because there wasn't enough DNA collected.
5. What is the source of the fluorescence? - The source of the fluorescence is the Ampicillin because it was the only variable added to the colored e.coli.
Conclusion:
To conclude, the hypothesis was strengthen because the number of colonies was increased in the LB+/Amp+ plates. As seen on the graph, were higher when the colonies found were higher. This was because IPTG was added and the resistance was created. The green fluorescent color showed this resistance. This statement can be proved through the graph shown (see Graph 1) that shows that all plates did produce a result of a changed e.coli.
Evaluation:
The limitations in this lab were that cross-contamination occurred through the pipets and the fluorescent color was limited in some plates which effected the overall group data. The data was very spread over the graph from 3 to 71 colonies and 6 to 142 transformations. These limitations effected the overall lab experiment.
Modifications:
Modifications can be made through this lab with more plates made to collect more e.coli.
Citations:
A packet was used as a resource and guide throughout the lab. This packet was titled "Transformation of E.coli with a Green Fluorescent Protein Plasmid" and it was created by EDVOTEK. This packet is labeled: EDVO-Kit # 223
