Annie and Brianna's IB Biology Lab

Transformation of E.coli with a Green Fluorescent Protein Plasmid:
Observation of an acquired phenotypic trait of green fluorescent protein exhibited by transformed bacterial cells.The process by which a bacterium takes up and expresses exogenous DNA through fluorescent proteins (gfp)in the transformed bacteria.

Introduction:
"Bacterial transformation is of central importance in molecular biology. Transformation is the process by which a bacterium takes up and expresses exogenous DNA, resulting in a newly acquired genetic trait that is stable and heritable. For transformation to occur, bacterial cells must be in a particular physiological state, referred to as competency, in which the bacterial cell was is made permeable to macromolecules such as DNA" (Edvotek). In this lab, the transformation of E. coli, an organism that does not naturally enter a state of competency, occurs with the help of a green fluorescent protein plasmid. Plasmids, which are self replicating extra chromosomal, double stranded circular DNA molecules, are found in many strains of bacteria. In this lab, the plasmid pFluoroGreen is used.The purpose of the plasmid, is to express fluorescent proteins in transformed bacterial cell. In summary, a strong promoter, a gene placed under the control of a DNA sequence, combined with an active RNA polymerase, allows for very high levels of gfp in transformed cells.The plasmid expresses the flourescnet protein because in addition to the antibiotic Resistance gene, it contains gfp gene known as pFlouroGreen.

Research Question:
Out of the four plates, labeled LB-, LB/Amp-, LB+ andLB/Amp+, which plate will successfully express the green fluorescent protein under the U.V. Light?

Hypothesis:
If the bacteria plate labeled LB/Amp+ is added with the pFlouroGreen plasmid, then the amount of transformations will be higher, than that of the plates labeled LB-, LB/Amp- and LB+. Consequently because the plate labeled LB/Amp+ has the the ampicilin resistance gene, as well as the green fluorescent protein gene.

Variables:
Independent Variable:

• pFlouroGreen, which is a gfp gene located in a plasmid, in addition to an antibiotic resistance gene, that produces the green fluorescent protein.

Dependent Variable:

• Amount of Bacteria Growth
  
• Amount of green fluorescent proteins, that glow under the U.V Light
  
• Rate of Transformation in colonies

Control Variables:

• Exact amount of bacteria colonies put into each tube of DNA+ and DNA-
  
• Exact amount of ampicilin put into the plates LB+ and LB-Exact amount of plasmid DNA , that is put into each peetry plate.


• Exact amount of Luria Recovery broth/ IPTG agar put into each peetrey plate
  
• Exact amount of Luria Recovery Broth put into the tubes containing the plasmid and bacteria concoction
• Exact amount of time that each tube is incubated in ice.


• Exact amount of time both transformation tubes spend at the temperature 42 degrees C for 90 seconds water bath. so the heat shock can facilitate the entry of DNA in bacterial cells.


• Exact amount of time the cells incubate for 0 minutes in a 7 degrees C water bath for a recovery period


• Amount of E. coli put into the peetry dishes.

Methods and Materials:

When doing this lap experiment it takes a long process, this experiment takes three days to be done correctly On the first day you prepare the agar plates agar and the E.coli cells ,these cells have to be incubated over night. As the plates are being made, the two striped plates don’t contain ampicillin and the two unstriped plates do contain ampicillin.On day one we also labeled all the tubes and plates that we used.On the second day of the lab, Take two micro centrifuge, label one “DNA+” and the other one “DNA-”,this has no pFG plasmid. Use a sterile 1ml pipette, add 250m ul(0.25ml) of ice cold CaCl2 solution to each tube. Take 2-4 colonies of E.coli using a toothpick from each plate and put it in each test tube. (Lb-, LB+, LB/Amp- and Amp+). Make sure you don’t take any agar when taking the E.coli from the source plates. Twist the toothpick vigorously and up and down in the cold CaCl2 to dislodge the cells. So that no cross-contaminations occurs, use a different toothpick for each test tube. Then close the tubes and shake or tap them to suspend the cells completely. Add 10m l of pFG (pFluoroGreen) to the “DNA+” test tube. Incubate the tow test tubes on ice for 15 minutes in a small beaker. Transfer both test tubes to a 42° C water bath for 90 seconds. After return both tubes immediately to the ice bucket and incubate for 2 minutes. Use a clean sterile 1ml pipette and add 250m l of Luria recovery Broth to each test tube, shake to mix. Incubate both test tubes for 30 minutes in a 37° C water bath for a recovery period. Put a thermometer in each water bath to make sure the temperatures are correct. While the tubes are incubating label the agar plates. Label one striped plate as “LB-”, and the other striped plate as “LB+”. Label one unstriped plate as “LB/Amp-” and the other unstriped plate as “LB/Amp+”. After the recovery period, remove the tubes from the water bath and place them on the lab bench, proceed on to plating the cells for incubation. Use a sterile 1ml pipette to transfer 250ml of recovered cells from the “DNA-” tube to the middle of the “LB-” and “LB/Amp-” plates. Spread the cells over the entire plate with a sterile inoculating loop. First spread the cell in one direction then spread the cells again in 90° of the first direction. Cover both plates and let the liquid be absorbed. Do this for approximately 15-20 minutes. Use another 1ml pipette to transfer 250m l of recovered cells from the “DNA+” tube to the middle of the “LB+” and the “LB/Amp+” plates. So that there is no contamination when plating, try not to put the lid down on the lab bench, lift the lid off the plate only enough to allow spreading. Use a different loop to spread recovered cells on each plate Stack the plates on top of each other and tape them together. Put your initial’s on the tapped set of plates. After the cell suspension is absorbed by the agar, put the plates in the inverted position, which means agar side on top. Then place the stack of plates into a 37° C bacterial incubation oven for overnight incubation. On the third day take the plates out of the incubator and look over the transformed plates. Darken the room and use a long wave U.V light to visualize the transformed cells that will glow as green due to the expression of the green fluorescent protein. Count the colonies of transformed E.coli in each plate. Finally compare your results with the other groups’ results.

Pictures of the materials used in the Lab:
















Qualitative Data:

Transformed Bacteria: Under the U.V. Light the bacteria was a fluorescent green, without the U.V. Light, the bacteria was a light brownish color

Odor of the Bacteria: Before and after the transformation the bacteria had a distinct smell

Picture of E. coli bacterium after transformation, under the U.V.Light
Quantitative Data:

Raw Data: Table 1: Data of each group in the class room and their respected information

Processed Data:
Table 2:

Table showing the 95% CI, Variance, Average,and Standard Deviation of the Raw Data , provided above. This is the mean/average, standard deviation, variance, and 95%CI for the amount E.coli colonies in the LB/Amp+ and the efficiency rate of the transformation of the bacteria

Graph 1:

Graph of Raw Data, Data of all the students information from the classroom

Graph 2: Graph of Raw Data with Error Bars


Conclusion:
Based on the data, the hypothesis, if the bacteria plate labeled LB/Amp+ is added with the pFlouroGreen plasmid, then the amount of transformations will be higher, was supported, because as seen on the graph, after the addition of the pFlouroGreen plasmid the amount of transformation's increased immensely in the plate labeled, LB/Amp+. In many of the groups the amount of colonies either doubled or increased even more. For example in Group 4, the amount of colonies they started with was 71; after the transformation, the amount of colonies more than doubled, to the amount of 142 colonies. In my own group, Group 7, we started out with 4 bacterium's and after the transformations we ended with exactly 8, in the plate labeled LB/Am+. This data, compiled with the data from the other seven groups, successfully reaffirmed the hypothesis, that was stated earlier. The data also answered the research question , which was, "Out of the four plates, labeled LB-, LB/Amp-, LB+ and LB/Amp+, which plate will successfully express the green fluorescent protein under the U.V. Light?". The answer that my partner and i were able to deduce, was that the plate labeled LB/Amp+, was the plate that successfully expressed the green fluorescent protein under the U.V. Light. To summarize this lab, the hypothesis and the research question were successfully answered and the amount of transformations were revealed to increase immensely in the plate that had ampicilin, which was supported by the raw data and the processed data.

Evaluation:

When creating this lab there were many steps to follow. There were many different mistakes made. Not only by our group, but by the other groups too, which means that everyone has different mistakes in their own labs. The first mistake made was with the broth, when mixing the broth it was not mixed equally which meant that there will automatically be errors in the results of the data. The amounts of broth in the groups were all different, some groups had more and some groups had less. This being said meant that the groups that had a more even amount a broth would have a higher transformational rate. Something else that could be wrong is the amount of DNA plasmid. When this is being done if not done right the groups will not have the right amount of plasmid DNA, and the E.coli will not be able to transform. There also may be various amount of cross contamination due to mixings of toothpicks or, and pipettes

Modifications:

Probably the most important modification that should be added to this lab, is to make sure that each group is not cross contaminating there experiment, due to the using of the same pipet. If each group successfully stayed aware of which pipet belonged to which tube, then the chances of cross-contamination would decrease immensely. Consequently because of that, the chances of having a more successful lab would in turn increase immensely as well. As stated in the evaluation section above, the amount of broth also caused some mistakes in the lab as well. By simply making one batch of the broth, everyone in the classroom could have a uniform amount of broth,which would consequently put everyone in the class on the same level/field. Having a responsible person to time the experiment, would also help this lab as well. Making sure that the amount of time, of the tube in the water bath, was properly calculated, would cause the lab to be more accurate. If the students had more time to prepare themselves the lab could be better. If we knew more about the lab then it would of bee easier for us to understand all the instructions for the lab.Simple modifications would greatly increase the accuracy of this lab and would also make it more enjoyable to the students.

Study Guide Questions and Answers:

1. On which plate would you expect to find bacteria most like the original transformed E. coli cells? I would expect to find it in the plate labeled LB-, because that specific plate is the control plate, which means it has nothing added to it, like the amipicilin or pFlouroGreen plasmid.
2. On which plate would you find only genetically transformed bacteria cells? I would expect to find the genetically transformed bacteria cells in the plate labeled LB+, because that specific plate consists of only the bacteria E. coli and the DNA.
3. What is the purpose of the control plates. Explain the difference between each and why it is necessary to run each? The purpose of the control plates, LB- and LB+, is to show the rate of transformation in the plates without the ampicilin. The control plates also are important because they would show which plate, the plate with DNA and the plate without, had more transformations.
4. Why would one compare plates LB/amp- and LB/amp+? One would compare the plates LB/amp+ and LB/amp -, to see the difference in rate of transformation in the plate with ampicilin and in the plate without ampicilin. To see the ampicilin's effect on the E.coli bacteria.

Work Cited:
Biotechnology Education Company (EDVOTEK) , (2003). Transformation of E. coli with a Green Fluorescent Protein Plasmid. Retrieved February 22, 2009, from EDVOTEK Web site: http://www.edvoteck.com/