Transformation of E.Coli with a Green Fluorescent Protein Plasmid by: Nahiomi Pagan and Mabeline Velez

Introduction: E.coli is a type of bacteria that does not enter a stage of consistensy and therefore it is a good organism to prove transformation. In order for this to happen, in this experiment we used Amphicillin, plasma membrane and Green Fluprescent protein. Amphicillin is an antibiotic that battles bacteria and this will make the plasmid DNA stronger through the process of transformation. The plasma membrane is very important beause it will bring the Green Fluorescent protein into the DNA in the cell making it transform and make new colonies that will have the Green Flourescent protein in it. The Green Fluorescent protein is also known as GFP and it will give the new colonies a green color that will be seen by placing it under an Ultra Violet light.

Purpose: The purpose of this lab is to have a better understanding about transformation. We will prove the method on how the transformation works. We will also know the parts of the cell that are used in order for transformation to take place.

Question: Which sample of E.coli cells will complete the transformation process successfully?

Variables: The independent variable of this experiment is the Luria Recovery Broth (LB), and the dependent variable is the Plasmid DNA.

Hypothesis: If the E.coli cell has a Plasmid DNA with LB and Ampicillin, then it will undergo transformation successfully.

Methods/Materials: It is important that one makes sure that all

the materials are ready and in place. This includes: 1ml Pipet, 0.25ml of ice cold CaCl2, Source plate of E.coli Cells, 2 Microcentrifuge tubes, Toothpicks, ice, beaker, 2 waterbaths, Inoculated Loops, and Lab bench.First make sure that you label one of your Microcentrifuge tubes "+DNA", this will be the one with the pFG Plasmid DNA; label the other tube "-DNA", which will be the one without pFG Plasmid DNA. Then use a sterile 1ml pipet to add 0.25ml of ice cold CaCl2 into each tube. Use a sterile toothpick to transfer 2-4 colonies from the source plate to each tube, but make sure that you use a different toothpick for each tube, do not use the same one for both. Also, avoid scraping the agar when getting the colonies. Twist the toothpick in the CaCl2 so that the cells will fall into the solution. Mix everything by tapping the tubes after closing them. Then to the tube labeled "+DNA" add 10ul of pFluoroGreen. Place the two tubes on ice in a beaker for 15 minutes. Then put both of the tubes in the WaterBath that is 42 degree Celsius for 90 seconds. Put the tubes back on the ice for 2 minutes. After, add 0.25ml of LB into each tube and mix it. Then place the tubes in the other waterbath which is 37 degrees Celsius for 30 minutes. While you are waiting label the 4 agar plates: one of the unstriped plates with "LB/Amp-", the other with "LB/Amp+", then one of the striped plates "LB-" and the other "LB+". After the 30 minutes have passed, remove the tubes from the waterbath and place them on the lab bench. Now take a sterile 1ml pipet and transfer the 0.25ml of cells from the tube labeled "-" into the plates that are labeled: "LB-" and "Lb/Amp-". Spread the cells with a sterile inoculating loop and cover the plates. Use another 1ml pipet and transfer 0.25ml of cells from the tube labeled "+" to the plates that are labeled: "LB+" and "LB/Amp+". Once again spread the cells with another inoculating loop and cover the plates. When finished, stack the plates on top of one another and place tape around them. Place the plates in the up right position so that the cell suspension can be absorbed by the agar. Then put the plates in a 37 degrees celsius Bacterial incubation oven for overnight. To see the results, darken the room and use an Ultra Violet light to see the cells which will glow green because of the pFG. Analyze results.

Data Results:

Analysis: Through the data that we collected we learned that transfromation happened because the plasmid will bring the new gene which is GFP, and it will transfer into the new colonies that have been transformed. Also by the number of colonies that we have counted we noticed that transformation did not occur in all of the colonies. In table 1 we noticed that the colonies that could be seen in the petri dish will not affect the number of transformatons that occur. In the graph we can also see that there are three groups that almost had the same amount of colonies and transformatipn but the rest of the groups were different. The reason for this could be because of human errors that took place during the mixing of the broth and ampicillin, the broth did not completely dissolve like it should have.

Qualitative Data: In the experiment, we were able to see the colonies that looked normal and had the normal gene that they usually have. In this experiment, the colonies that changed and were turned green looked different from the rest of them. When we placed the E.coli that had the Fluorescent gene under the Ultra Violet light we were able to see that the bacteria was glowing green. Even without the light we were still able to see the E.coli which had a different color and we could see that the colonies were green.

Quantitative Data: When looking at the quantitative data, we were able to see that while doing this experiment we have had made some human errors, which is the reason why everybody's results were different. This data also showed the numbers of colonies that each group had during this experiment.

Safety Considerations: It is very inportant to be cautious during this experiment and safe. It is neccessary for one to wear gloves at all times during this experiment because one will be dealing with bacterias. Always make sure that the materials are clean and do not fool around with any of the solutions that will be around. It is essential that one washes one's hands before and after the experiment. When finished with the experiment, throw away all materials used into the red bag that will be around for contaminated materials. After throwing away your gloves wash your hands.

Evaluation: This experiment allowed the students to have a better idea about transformation and how it's process worked. One of the limitations of this experiment ocurred on the first day of the experiment. We did not finish on time which means that we satyed behind; next time we will use a better sense of timing ourselves so that we can be able to finish on time. Also keeping everything sterile is very important for this experiment to be successful. Following directions is a good way of being able to do everything right and end up with good results.

Modifications: These modifications will make the experiment better to do and even easy. One major thing is keeping everything clean and sterile. Making sure that the materials used for one solution is not used again for another solution is crucial for this experiment. A simple mistake can ruin the whole experiment because of cross-contamination. Another thing is to always keep the petri-dishes closed because the bacteria from the air or anything that is surrounding it can go in and ruin the whole experiment.

Conclusion: This is a good experiment for students to learn first-hand how transformation works. When a cell undergoes transformation, the DNA has to prepare itself and divide itself so that when it is divided, the other half will have the same characteristics and abilities as the other. So the "mother" which is the original DNA, is the same as the "daughter" , which is the replica of the original DNA; they both serve the same function. This is the reason why in this experiment we saw many cells that were the same and why they were all green, they went through transformation and made more of themselves. And so our hypothesis was correct; the dishes with the plasmid DNA produced more E.coli than the other dishes that did not have plasmid DNA. The transformation went successful.

Bibliography:

(97). EDVOTEK-The Biotechnology Education Company. Retrieved February 25 ,2009, from www.edvotek.com