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

1/5/14-1/10/14

We're finally back in school after our winter break. The semester is ending soon, and midterms are coming up. These next couple of weeks will be very busy, full of review for semester exams. I'm actually most worried about English, since we're tacking a practice AP Exam as our midterm. My break was enjoyable, but nothing particularly exciting happened. It was just lots of much-needed rest and relaxation. The quiz we took on Monday told me I was neither a morning person nor an evening person; I though this was surprising, since I'm definitely more alert later in the day. This has shown me that these quizzes aren't always reliable.

12/7/14-12/13/14

Mammal Lactation

Lactation in mammals is a positive feedback loop, because milk is continually produced until the offspring stops breastfeeding. The response increases the stimulus. It is not negative, because it does not seek to limit a detrimental stimulus. In this case, the stimulus is the baby suckling, which results in signals being sent to the hypothalamus that then releases oxytocin, which in turn results in continued milk production. This does not end until the baby is done suckling.

12/1/14-12/5/14

This week, we spent most of our time on the diffusion and osmosis lab, with minimal notetaking or homework. I thought this was genuinely interesting; we had to apply our knowledge in the labs, reinforcing the material even without a lecture or a powerpoint. I wouldn't want to jump into this feet first, though; that is to say, I prefer having a solid foundation from notes and lectures before doing a lab. It won't be long before winter break, which will be a much needed respite from all my schoolwork; however, it won't be long after that until we have to take midterms. I'm going to look through my "5 Steps to a 5" book over break to review older material and get a glimpse of what lies ahead, so I can do well come the end of the semester.

Diffusion and Osmosis Lab Reflections

Investigation 1 dealt with surface area to volume, and why cells are as small as they are. We hypothesized that the 1cm cube would have the highest diffusion rate since it had the highest ratio of surface area to volume. The results supported our hypothesis, and the 1cm cubed was the only one that displayed full penetration. Cells remain small because a higher surface area allows for more cellular activity, and having a high ratio of surface area to volume provides an optimal surface area in a compact space.

In investigation 2, we created models of cells using dialysis bags and filled them with a solution (in our case, 10ml of 1M sucrose), and placed them into a beaker containing another solution (for our group, 100ml of 1M glucose). We weighed the models before placing them in the beakers and then again, after removing them after a 30 minute soak. For us, the cell gained 12.9% more of its' original mass. The underlying concept behind this investigation was tonicity; molecules will diffuse down a gradient until equilibrium is reached. Our cell gained mass, suggesting that molecules flowed into the "cell" from the solution; thus, this suggests that the solution was initially hypotonic.

Investigation 3 involved observing the cells of an onion through a microscope. First, we looked at the cells without placing water on them; then, we exposed the cell to both distilled water and saltwater. The distilled water resulted in water flowing into the cell, as the concentration in the cell was lower. When exposed to saltwater, however, the cell lost water and shriveled, as the concentration of water in the cell was higher than in the saltwater. This exemplifies both the effects of differing tonicities, and the fact that the amount of free water is more important than the total mass. There might have been just as many water molecules in the saltwater, but many of them were unavailable due to being bound to salt molecules, thus resulting in a hypertonic solution.

10/5/14-10/10/14

The most memorable activity for this week was definitely the BLAST lab. It was a bit confusing at first, due to the expansiveness of the phylogenetic trees, but eventually I understood it. It was interesting to see how biotechnology can be applied to criminal justice; the two seem like very unrelated fields, but they are really complementary. I also attended the lecture at Whitney Lab on bioluminescence in marine organisms. While it was definitely engaging, I'd like to see more applications of the research besides science for science's sake. I remember hearing something about inserting fluorescent genes into other organisms allowing us to see them more easily, but I feel like that could have been elaborated on more.

9/29/14-10/3/14

This week wasn't particularly stressful; I didn't really have trouble with anything in class and understood everything fairly well. We got our assignment to campaign for a specific piece of evidence that supports evolution. My group is doing embryology. It seems like a lot of work, but it's also interesting, and the competitive aspect is certainly a good motivator. I can't help but wish I'd gotten DNA or fossil records instead, though. I'm more familiar with those than with embryology, so I'll have to conduct more research. Our first FRQ is coming up soon; I'm not worried about it, but I certainly don't think I can just breeze through it either.