-from biobuilder
put in the plates
talk about why there was no growth.
4-1, 4-2, mm294(this last strand was one whe just found in the fridge, and we wanted to see what would happen with it.
after it didn't work we took 4-1, 4-2, and mm294 and spread them on lb agar.
put all of this on the project notebook.
This lab was performed twice. The lab was done with the prescribed 4-1 and 4-2 and an additional strand the was found in the school fridge. There was some curiosity as to it would grow and change when it was made competent. The first time there was no growth on the LB amp plates from any of the strands, and while this could
1 December 2016
Transformation Lab with 8th graders from Peabody School
Peabody School is a Pre-K - 8th grade private school located in Charlottesville Va.
On this day, the 8th grade biology class came to Renaissance School to work perform a transformation lab. The children were split into three groups, Acree, Nico, and I each took our own group. Each group was given a strain of E- coli and pGRN and pPRL plasmids. My group was given the 4-2 strain, Nico’s group with the 4-1 strain, and Acree was given the m294 strain, which we had found in our refrigerator from prior experiments. Each group was given their own strain of bacteria to transform to save on the number of plates needed. Before we transformed, we made the cells competent.
The procedure is below.
What a Colorful World Transformation
Part 1: Preparing bacterial strains for transformation
Neither of these E. coli strains will take up DNA from the environment until they are treated with a salt solution that makes their outer membrane slightly porous. The cells will become “competent” for transformation (ready to bring DNA that’s external to the cell into the cytoplasm where the DNA code can be expressed). The cells will also become fragile. Keep the cells cold and don’t pipet them roughly once you have swirled them into the CaCl2 salt solution.
1. In advance of lab today, a small patch of each strain was grown for you on an LB agar petri dish.
2. Label 3 small microcentrifuge tubes with the bacteria strain you are working with.
3. Pipet 200 µl of CaCl2 solution into each microcentrifuge tube and then place the tubes on ice.
4. Use an inoculating loop to scrape up one entire patch of cells (NOT including the agar that they are growing on) and then swirl the cells into its tube of cold CaCl2. Gently flick and invert the microcentrifuge tube, then return it to your ice bucket.
5. Keep these competent cells on ice while you prepare the DNA for transformation.
Part 2: Transforming competent cells
The cells you’ve prepared will be enough to complete a total of 6 transformations. You will transform both the purple-color generator and the green-color generator into one of the three bacteria strains. You will also use the last bit of competent cells as negative controls for the transformation.
1. Retrieve 3 aliquot of each plasmid for a total of 6 samples (3x pPRL and 3x pGRN). Each aliquot has 5 µl of DNA in it. The DNA is at a concentration of 0.04 µg/µl. You will need these values when you calculate the transformation efficiency at the end of this experiment.
2. Place the microcentrifuge tubes (pPRL and pGRN) in the ice bucket.
3. Flick the tube with the competent bacteria strain and then pipet 100 µl of the bacteria into the tube labeled pPRL. Then pipet 100 µl of the competent bacteria strain into the tube labeled pGRN. Flick to mix and then store them, along with the remaining volume of competent cells, on ice.
4. Let the DNA and cells sit on ice for 5 minutes. Use a timer to count down the time.
5. While your DNA and cells are incubating, label the bottoms (not the tops) of the petri dishes you will need. The label should indicate the strain you’ve used (4-1,4-2,or M294) and the DNA you’ve transformed them with (pPRL, pGRN, or no DNA control).
6. Heat shock all of your DNA/cell samples by placing the tubes in a 42 degree waterbath for 90 seconds exactly (use a timer). This step helps drive the DNA into the cells and closes the porous bacterial membranes of the bacteria.
7. At the end of the 90 seconds, move the tubes to a rack at room temperature.
8. Add .5 ml of room temperature LB to the tubes. Close the caps, and invert the tubes to mix the contents.
9. Using a sterilized spreader, spread 250 µl of the transformation mixes onto the surface of LB + ampicillin agar petri dishes.
10. Plate the remaining volumes of transformation mixes on LB plates to show the effect of antibiotic selection on outcome.
11. Incubate the petri dishes with agar side up at 37 degrees overnight, not more than 24 hours.
Results
Acree’s group, E. coli strain M294
pPRL: Resulted in one colony, with pinkish hue. We believe that it was pink because that’s as far as the chassis was able to synthesize the violacein protein or the bacteria needed a supplementary protein in order to synthesize.. Transformation efficiency = 10
pGRN: Acree had 6 colonies, with a dark green hue. TE = 60
Sana’s group, E. coli strain 4-2
pPRL: 2 colonies, extremely dark purple hue. TE =20
pGRN: 3 colonies, very dark green hue. TE=30
The purple and green were very similar in color.
Nico’s group, E. coli strain 4-1
pPRL: a lawn of bacteria with no color at all. TE= 0
pGRN: no growth. TE = 0
During Nico’s transformation he accidentally diluted his pGRN plasmid with 20 microliters of CaCl2. This may have resulted in his lack of transformation with the pGRN plasmid. However, his pPRL plasmid clearly transformed since there was growth on the an AMP plate but there was no color. Perhaps, his strain is unable to synthesize the violacein protein at all which resulted in the no growth on the pGRN plate. An additional transformation needs to be performed to determine if strain 4-1 is able to synthesize the protein.
December 5, 2016:
From the transformation performed Dec. 1st, the 4-1 strain that was transformed with the pPurple plasmid showed growth on the plate, but there was no visible color change in the bacteria. A sample from this plate was transferred to a new LB/Ampicillin plate to further examine these results by growing the bacteria again to ensure that the growth was actually transformed bacteria lacking color and not bacteria growing on a plate with inactive Ampicilin. The sample grew on the new plate, but there was still not a change in the color of the bacteria. Because the 4-1 strand did not have any growth when it was transformed with the pGreen plasmid, it was not re-plated. However, both the 4-2 and the M294 strains had growth for both plasmids, so they were both plated again on LB/Amp plates to grow a population of bacteria in order to complete a miniprep procedure to isolate plamid DNA again since we were out of the original plasmids.. The M294 with pPurple and pGreen and the 4-2 with pPurple and pGreen plates all showed significant growth, but the two plates that included the PGreen plasmid (one with the M294 strain and one with the 4-2 strain) exhibited a visible change in color from when they were originally transformed. This color was similar to the color of the 4-2 strain that was transformed with pPurple; the green appeared to be less of a true green, which contrasted with the original transformation that showed a dark, clear green, while the 4-2 strain with pPurple had similar coloration to the originally transformed plate. Lastly, the M294 strain with pPurple maintained a very similar color to the single colony on the original plate. It had a salmon-colored hue and had significantly greater growth.
We no longer had a supply of either plasmid to transform strain 4-1 to determine the bacteria’s ability to synthesize the protein. Because of this, a mini prep procedure was conducted in order to isolate the DNA so that another investigative transformation could be performed. First, a sample of each of the transformed 4-2 strains (one with PGreen and one with pPurple) were grown in liquid Luria Broth, in addition to the M294 strain with pPurple. After the three tubes were incubated overnight, a miniprep procedure, which isolated the DNA from the cells, was performed. The collection of plasmid DNA from the mini prep will make it possible to conduct another transformation
