Transformation Lab Report
Abstract
The purpose of this lab was to insert genes that would make E. coli resistant to ampicillin and to glow. Genetic transformation is an active uptake of free DNA by a bacterial cell and the incorporation of the genetic information. In this experiment plasmids, are inserted into a host E. coli cell. The lux operon is an operon that contains a gene for luciferase and a portion of the gene also has a resistance for ampicillin. This gene codes for enzymes that produce luciferins. These are what make the cell illuminate. In this lab we performed a genetic transformation of E. coli cells.
Introduction
The purpose of this experiment is to observe the transformation of bacterial cells. The basis for the knowledge of transformation began with Frederick Griffith’s experiments with mice in the 1920’s. The scientist found that the one strain of S. pneumoniae that made smooth colonies causes pneumonia and ultimately death for the mice while the rough looking colonies strain was harmless. However, when he mixed dead cells of the smooth strain with the rough strain, the mice became ill just as they did with the living smooth cells. Two of the harmless cells together made a harmful impact. Griffith proposed that the rough cells obtained the DNA from the smooth cells, therefore transforming them. The cells now had new attributes (genetic diversity). This is where the idea of transformation came from. In this lab, we test to see if E. coli cells can obtain DNA (plasmids) to have a new genetic make up and therefore resistances (from ampicillin, kanamycin, ability to glow).
Methods
The methods we used are the same methods that are used in laboratories that perform these tests in order to use the results to modify genes of different organisms. First we started by marking one of our 15- ml tube “+plasmid” and the other”-plasmid”. The DNA was only added to the “+plasmid” tube. We will be referring to each tube by their respective signs, either - or +.
After the tubes were marked we then began to add .25 mL of ice-cold calcium chloride to each tube. We then proceeded to place both tubes on ice. After both tubes were in the ice we placed colonies of E. coli into the + tube with an inoculating loop. The approximate area of the colonies were about half of the top of an eraser. When the inoculating loop was placed in the tube it was spun around to insure we dislodge the cell mass. Holding the tube up to a light verified that the cell mass did indeed fall off into the tube.
Immediately after we suspended the cells by pipetting in and out with a transfer pipette that was sterilized. We then verified that there were no cell masses within the pipette by holding it up the a light source. After all of this was completed the + tube was put back in the ice.
A mass of cells was then transferred into the - tube and suspended multiple times before being placed back within the ice. We then plasmid DNA to the + tube and put it through cell suspension again. After being placed back on ice, both tubes were incubated for 15 minutes. During the process of incubation we labeled our petri dishes. The labels include our group number, the date, if it was -plasmid with ampicillin, kan and nothing. The same happened with the other three petri dishes except they were labeled with a +plasmid rather than -plasmid.
After the 15 minute incubation period the tubes were then heat shocked. In order to heat shock the tubes, we placed them into water that was 42 C for 90 seconds. As we held them in the water we spun them around or ‘agitated’ them. This was one of the most crucial steps and has to be performed with precision.
After the heat shock was complete, .25 mL of Luria broth was added to the cell solutions and agitated by added glass beads. They then sat at room temperature for 5-15 minutes. After the incubation period we followed certain procedure in order to spread the cells in the +plasmid tube onto the +plasmid plates and do the same with the cells in the - tube.
Prior to adding the cells, make sure to put 4-6 glass beads in each petri dish to be used later in the stirring process.Using sterile pipettes add .10 mL of the cells to their corresponding petri dishes(+plasmid tube to +plasmid petri dish and so forth). Then begin to stir the petri dishes for 2 and a half minutes. After 2 and a half minutes have passed, discard of the glass beads how ever your instructor tells you to. Once your instructor has advised you all how to incubate your dishes until the next day, this is when you will record your results.
[See citations for resource on our methods]
Results/Data: Include pictures, data tables
/ LB Ampicillin Plates / LB Kanamycin Plates / LB Plates /
+ plasmid
- plasmid
+ plasmid
- plasmid
+ plasmid
- plasmid
Color of colonies
Plasmid 1
no colonies*
lawn*
no colonies
no colonies
lawn
no colonies
n/a
Plasmid 2
no colonies*
lawn*
no colonies
no colonies
lawn
no colonies
n/a
Plasmid 3
no colonies*
lawn*
no colonies
no colonies
lawn
no colonies
n/a
* : the “- plasmid” and “+ plasmid” plates were switched on accident. That is why we have conflicting results.
Does the plasmid below contain
an ampicillin-resistance gene?
a kanamycin-resistance gene?
a green fluorescent protein gene?
Plasmid 1
yes
no
no
Plasmid 2
no
no
no
Plasmid 3
no
no
no
The film you may see is most likely condensation. There were lawns on the amp LB- and LB+ plates, though.
Below, you can see the Amp LB - (which was actually Amp LB+) glowing with a lawn and a few colonies.
Discussion
We found that our E. coli cells were only transformed with the plasmid for ampicillin-resistance. The lack of results most likely due to human error when transferring the E. coli to the + and - plasmid tubes and heat shocking them. Also, we have seen that across our class’ experiments, there was a lack of results with the kanamycin plates, proposing a bad stock of kanamycin-resistant plasmids. We should have been able to tell which plasmid was where but all we found was that the E. coli was transformed to be ampicillin resistant and that bacteria was present (seen in the LB plates). Additionally, we were surprised to find that our “- plasmid” LB Ampicillin plate had a lawn while our “+ plasmid” LB ampicillin plate (which was supposed to have a lawn) did not. These opposing results are attributed to the switching of “+/-” lids.
Laboratory Questions:
Record your results and conclusions using the organizational method you devised in the Pre-laboratory Inquiry Activity.
Did you observe what you expected to? If not, how would you explain your observations?
We did not observe what we expected to as far as which plates would have growth and which would not. However, we believe this is because we got the lids matched up. We saw a great amount of growth on a petri dish that should’ve had none and none of a petri dish that should’ve had a great amount of growth. We know that we followed the procedures correctly therefore we are pretty sure that it was a mislabeling error.
The results from these three experiments are described as a, b, and c. Something has gone wrong with each of these transformations. Use the controls to figure out what has gone wrong.
Results from Experiment 1:
All plates did not have a lawn or colonies
Results from Experiment 2:
Both positive and negative KAN plates were clean, the other had lawns
Results from Experiment 3:
Colonies on the ampicillin plates, lawns on the LB plates and clean plates on KAN
Having a way to measure transformation efficiency helps in discussing results or in comparing transformations that were not done at the same time. Transformation efficiency is expressed as the number of transformed colonies (in this case those that are antibiotic-resistant) per microgram of plasmid used in the transformation.
Figure out how you would calculate transformation efficiency (i.e. number of colonies/ microliter of plasmid used). You used 10 microliters of plasmid at a concentration of 0.005 micrograms/microliter.
Conclusion
We have learned the process of making bacterial cells transform as well as the abilities they can have after. This process, although proved difficult for our group, is replicated in nature all the time. This knowledge of how bacteria can transform helps us better come up with plasmids for resistance to harmful diseases. The ease with which an efficient plasmid can be obtained is integral for a successful final product. We want to create a solution or vaccine that can help people. The sufficiency of this vaccine depends on our ability to speed up the transformation efficiency of the plasmid. Our knowledge from this lab about how the process of transformation takes place will help us with that.
Additionally, we have learned that you must read and reread the directions, measure and remeasure, and not skip any steps. One of the reasons our results were wrong was because we forgot to cool the tip of the applicator for E. coli or put the solution in the wrong plate.
Resources
Carolina Transformation for AP Biology. (n.d.). Retrieved December 17, 2014, from https://echo.newtechnetwork.org/sites/default/files/new_uploads/20141211/_1418316053_Transformation Lab.pdf
Reece, J. (2012). Campbell biology: Concepts & connections (8th ed., pp. 180-230). San Francisco, CA.: Benjamin Cummings.
Wootton, K. (Director) (2014, December 1). Bacterial Transformation. AP Biology Class. Lecture conducted from New Tech High @ Coppell, Coppell.