LAB:
Transforming E. Coli Cells With Fluorescent Plasmid Protein
BY:
Marshawn Henry
INTRODUCTION:
Bacterial transformation is of central importance in molecular biology. In order for transformation to occur, the bacteria must be in a state known as 'competency'; which is when the cell is made permeable to DNA. In this lab, we will be able to transform E. Coli which normally doesn't transform on its own with the aid of a green fluorescent protein plasmid called pFluoroGreen.
HYPOTHESIS:
The amount of transformations will be higher in the plate labeled LB/AMP+ because the pFluoroGreen plasmid was added.
RESEARCH QUESTION:
Out of all four plates, which will be most successful showing the fluorescent protein under the u.v. lighting?
INDEPENDENT VARIABLES:
- The gfp gene pFluoroGreen which is located in the plasmid.
DEPENDENT VARIABLES:
- Transformation rate among the bacteria colonies
- Bacterial growth
- Amount of green fluorescent proteins
Materials:
Methods:
Start off by labeling one micro centrifuge tube +DNA (contains pFG plasmid DNA) and another -DNA (without pFG). Use a sterile 1 ml pipet to add .25 ml of ice cold CaCI2 solution to each tube. Now use a sterile toothpick to transfer several colonies of E. Coli to each tube by twisting the toothpick vigorously in the CaCI2 solution. Tap on tubes to suspend cells completely. Add .010 ml of pFG to tube labeled +DNA.
Now place both tubes on ice for 15 minutes. The, store tubes at 42 degrees Celsius for 90 seconds and return to ice immediately for two minutes. Add .25 ml of Luria recovery broth using a sterile pipet. Next you incubate the cells for 30 minutes in a 37 degree Celsius water bath, this is known as the recovery period.
Label four agar plates while cells are recovering. Label one unstriped plate LB/Amp- and another LB/Amp+. Then label one striped plate LB- and the other LB+. After recovering; place tubes on lab bench. Now use a sterile 1 ml pipet to transfer .25 ml of recovered cells from the tube labeled -DNA to plates labeled LB- and Lb/Amp-. Spread cells using the inoculating loop. Using a different pipet, transfer .25 ml of cells in the +DNA tube into plates labeled LB+ and LB/Amp+. Cover all plates and allow liquid to be absorbed for approximately 20 minutes.
Place all plates on top of each other in the upright position to allow the cell suspension to be absorbed by the agar. After, plates will be placed in a 37 degree Celsius bacterial incubation oven overnight in the 'inverted' position. To view plates, darken room and use a long wave U.V. light to visualize transformed cells that glow green due to the fluorescent protein.
QUESTIONS:
- On which plate(s) would you expect to find bacteria most like the original non-transformed E. coli cells? - I would expect LB- to be most like the original E. coli cells because it had no pFG or ampicilin to aid the E. coli to transform.
- On which plate(s) would you find only genetically transformed bacterial cells? - I believe the plate labeled LB+ would only be genetically transformed because it consists only of the E. coli and the DNA.
- What is the purpose of the control plates? - The purpose of control plates is to
- Why would one compare plates LB/Amp- and LB/Amp+? - LB/Amp- and LB/Amp+ would be compared to see the difference the ampicilin makes on the transformation of the E. coli.
DATA:
GROUP #OF COL. #OF TRANS.
1 15 30
2 3 6
3 28 56
4 71 142
5 5 10
6 28 56
7 4 8
8 29 58
CONCLUSION:
In conclusion, my hypothesis was proven to be true. The plate labeled LB/Amp+ had an increasing number of transformed colonies compared to the other plates. This lab proves that using pFG and ampicilin will aid E. coli cells reach competency and therefore transform into more colonies or bacteria.
