Transformation Lab Writeup

Transforming E. Coli With GFP and Ampicillin Resistance

Introduction

Background

Through the process of transformation, bacteria are capable of taking up DNA from their environment (in this case, through exposure to a plasmid containing the desired genes). This gives the bacteria new genetic information that is stable and can be passed down to future generations. In this experiment, we transformed  E. Coli with pFluoroGreen, which contains genes for ampicillin resistance and Green Fluorescent Protein (GFP). Furthermore, we exposed some of the cells to IPTG, which is required for GFP to be expressed. Thus, the experiment demonstrates differential gene expression.

Hypothesis

If E. Coli is exposed to both IGTP and pFluoro-Green, then fluorescent bacteria with resistance to ampicillin will grow.

Materials and methods

Materials

• BactoBeads E. Coli GFP Host
• pFluoroGreen plasmid DNA
• Ampicillin
• IPTG
• CaCl2
• Growth Additive
• ReadyPour Agar
• Recovery Broth
• Petri plates (small and large)
• Plastic tipped micropipettes
• Toothpicks (sterile)
• Inoculating loops (sterile)
• Microcentrifuge tubes
• Automatic micropipettes and tips
• 37 degree C & 42 degree C water baths
• Thermometers
• Incubation Oven (37 degrees C)
• Ice
• Markers
• Bunsen burner/hot plate
• Gloves
• Long wave UV light

Methods

We began by labeling our + and - microcentrifuge tubes. Then, we transferred CaCl2 solution into the - tube, and then added about 15 colonies of E. Coli to the same tube. We vortexed the solution, and noticed that it was still very clear. So, we added more colonies and vortexed again. It was still clear, but less so; thus, we continued by extracting half of the 500 microliter solution and placing it in the + tube. Afterwards, we added pFluoroGreen to the + tube, while leaving the - tube alone. We iced both tubes for 10 minutes, placed them in a 42 degree C bath for 90 seconds, and then iced them for 2 more minutes. We then added 250 microliters of recover broth to each tube, and then incubated them for half an hour in a 37 degree C bath. While we waited, we labeled our four agar plates as follows: -DNA,-DNA/+Amp,+DNA/+Amp,+DNA/+Amp/+IPTG. After recovery ended, we transferred the solutions to their appropriate plates and spread the cells over the plates using the inoculating loops. After 5 minutes, we stacked and taped our plates together, and incubated them overnight. Finally, we placed them under UV light in the morning and observed our results.

For specific steps refer to: http://www.edvotek.com/site/pdf/223.pdf

Statistical Methods

We are going to calculate transformation efficiency and then plot the class’ data on a graph, establishing standard deviations.

Results

Dependent variable: Number of E. Coli colonies present and whether or not they were fluorescent/ampicillin resistant

Independent Variable: Exposure to the plasmid and/or IPTG

Confounding Variables: All E. Coli extracted from same plate, all plates incubated for same amount of time, all measurements that needed to be equal in both samples kept equal (example: both had 250 microliters of recovery broth added), sterility maintained

Replication/Sample Size: We did not repeat our trials, but we will be using the class’ data, so we have over 20 samples.

Controls: The -DNA and -DNA/-Amp plates served as controls

Data: Measurements still have to be taken; none of our bacteria expressed fluorescence

Data Analysis: Measurements must be taken and data must be shared in the class before we can analyze data. While none of the bacteria expressed fluorescence, we did have some colonies grow-albeit few.

Conclusions

None of our bacteria displayed fluorescence. At first, I thought it might have been that they simply required more time to incubate. However, even after inspecting the plates under the UV light again after some time, we had no glowing colonies. Much of the class lacked glowing bacteria too; in fact, I only saw one plate with glowing bacteria. Perhaps the E. Coli simply failed to integrate the DNA from the plasmids. Human error is also a likely cause-in our case, I think we might have transferred the colonies from the plate to the tube improperly. If we had the opportunity to repeat this experiment, I would make sure to follow the instructions more carefully and pay special attention to the initial transfer of the E. Coli into the microcentrifuge tube. Adding more colonies might have helped our chances of having some of the bacteria express fluorescence, too.

Literature Cited