Transformation Efficiency Calculation

Number of transformants per ug.
(Number of Transformants / ug of DNA)
X
(final volume at revovery ml/volume plated ml)
The final volume at recovey is 0.50 ml
The volume plated is 0.25ml

still in progress of undating..........................

Picture of the lab (1)

This is the picture of the transformed colonies of bacteria from all eight groups on the LB/Amp+ plate. The one with the most colonies is mines.

Picture of the lab (2)

Equipments used during the transformation lab.

Title

Transformation of E.coli with a Green Fluorescent Protein Plasmid.

Research Question

The effect of plasmid DNA on the transformation of the E.coli bacteria with a Green fluorescent Plasmid measure by the colonies of bacteria formed and calculated by the efficiency rate formula.

Introduction

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 bacterial cell walls is made permeable to macromolecules such as DNA. E.coli can artificially be made competent when treated with chloride salts of the metal cations calcium, magnesium, and rubidium. In addition, abrupt transitioning between heat and cold can induce competency. It is believed that metal ions and temperature changes affect the structure an permeability of the cell wall and membrane, allowing DNA molecules to pass through. Transformed cells will grow on selective medium that contains antibiotic. To ferry foreign genes into bacteria, plasmids are usually used. Plasmids are self replicating extra chromosomal, double stranded circular DNA molecules found in many strains of bacteria. Only bacteria cells that take up the plasmid will survive selection on ampicillin agar plates and will produce green fluorescent colonies which will be visible under the long wave U.V. Light. The transformation efficiency rate for E.coli with a green fluorescent protein plasmid would be higher under the condition "LB/Amp+" due to the facts that plasmids are used to ferry foreign genes into bacteria and transformed cells will grow on selective medium that contains an antibodies.

Independent Variable

DNA plasmids

dependent variables

The express of the pFluoroGreen in bacteria

Methods and Materials

This experiment is a three day experiment. On day one, prepare the agar plates and the E.coli cells plates( which will be incubated over night in an incubation oven at 37C). When preparing the agar plates, the two striped plates will not contain ampicillin and the two unstriped plates will contain ampicillin. put the CaCl2 solution and the pFluoroGreen on ice (They should be keep in the ice so it stay coold at all times). On day two of the lab, transform cells and plate for overnight incubation. Take two micro centrifuge, label one “DNA+” and the other one “DNA-”(this will contain no pFG plasmid). Use a sterile 1ml pipette, add 250m l(0.25ml) of ice cold CaCl2 solution to each tube. Transfer 2-4 colonies of E.coli from the source plate to each test tube using a toothpick. Be careful do not scrape up any agar when taking the E. coli from the source plate. Do not wipe the bacteria cells on the side of the micro centrifuge. Twist the toothpick vigorously and up and down in the cold CaCl2 to dislodge the cells. To prevent cross-contaminations use different toothpick for each test tube. Then close the tubes and tap or shake the tubes to suspend the cells completely. Add 10m l of pFG (pFluoroGreen) to the “DNA+” test tube. Incubate the tow test tubes on ice fro fifteen minutes in a small beaker. Transfer both test tube to a 42° C water bath for 90seconds. Then 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 and mix by shaking. Incubate both test tubes for 30 minutes in a 37° C water bath fro a recovery period. To make sure that the temperature is correct, put a thermometer in each water bath. While the tubes are incubating label the agar plates. Label one striped plate as “LB-”, and the other striped plate as “LB+”. Labe 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. Then proceed on to plating the cells fro incubation. Use a sterile 1ml pipette to transfer 250m l 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 allow the liquid to be absorbed(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. To avoid contamination when plating, do not set the lid down on the lab bench, lift the lid of the plate only enough to allow spreading. Use a different loop to spread recovered cells on each plate. Be careful to avoid gouging the loop into the agar. Stack the plates on top of each other and tape them together. Put your initial son the tapped set. After the cell suspension is absorbed by the agar which takes around 20 minutes, flap the stake of plates over so that the agar side is on the top. Then place it into a 37° C bacterial incubation oven fro overnight incubation. On day three , observe transform ants and controls. 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. Dispose all material porperly in a red hazard bag, this includes the bacteria plates. Compare result with other groups.

Raw Data-chart1

This is the transform bacteria counts that was compare in class among the eight group of experiments on the third day of the lab.

Analysis-chart2

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.

Analysis-graph

This graph represents the 8 lab groups' result. The purple represents the bacteria colonies and the pink is teh transformed colonies.

Conclusion

In conclusion, my hypothesis is supported by the experiment that the LB/Amp+" will more pFG. in fact, it is the only plate of bacteria that transformed. E.coli will transform under the condition that it contain an antibodies and plasmid DNA. It is due to the plasmid that the bacterias are able to take in the pFluorescent Green and resist against antibodies. And base on the other plates, we can assume that the antibodies with out plasmid DNA will not cause transformation in bacteria it will cause the death of the bacteria. The data that is collected among the eight groups are very diverse in number and this lowers the reliability of the numbers. My group have 71 colonies of transformed E.coli. the highest result among the eight groups.

Evaluation and Modifications

There are mistakes make by different group during the lab that lead to the diverse result. The first human error identified is the broth. When the broth is mixed, it was not mixed equally. Some part have more and some have less, so the group with the more evenly distributed broth will probably have a higher transformational rate. Another human mistake can be cause by the amount of DNA plasmid. If the equipment is not used properly, the group will have less plasmid DNA for the E.coli to transform. Other that have transformed bacteria on other plates is due human error of cross contamination. Either they use the same loop for all the plates or it is because they misplace their lid somewhere and then put it back on the plate.
This lab will be better if the students are able to do the pre-lab preparation themselves. Also, it can be a self experiment after knowing all the background information that is necessary for the lab instead of a follow the instruction lab, it will be more interesting.

Work Cited

Biotechnology Education Company (EDVOTEK) , (2003). Transformation of E. coli with a Green Fluorescent Protein Plasmid. Retrieved February 25, 2009,
EDVOTEK Web site: www.edvoteck.com