Background:
Bacteria can transfer DNA by using either transformation or conjugation. Transformation and conjugation are different in the aspect of how the bacteria receives the DNA. In transformation the bacterium grabs any extra DNA from its environment while in conjugation one bacteria inserts its DNA into another bacteria.
In this lab we will be transforming E.Coli bacteria with ampicillin resistant and glowing plasmids. You will have 2 +DNA plates. One with AMP to see if they have been successfully transformed and a plate containing IPTG which will help induce the lac operon which will make the transformed bacteria glow under U.V. light.
*anyone allergic to penicillin, ampicillin, kanamycin, or tetracycline should not participate*
Agar plates should already be prepared. Aseptic techniques must be used and proper PPE must be worn.
Materials:
Pre-lab questions:
1. On which plate(s) would you expect to find bacteria most like the E. coli on the
source plate? Explain.
I would probably expect to find bacteria like the E.Coli on the source plate -DNA. I would expect that one since no plasmid would have been added and there are no antibiotics being applied; it would be the same bacteria from the beginning.
2. On which plate(s) would you find only genetically transformed bacterial cells?
Why?
You would find strictly genetically transformed bacterial cells on the +DNA/+AMP/+IPTG because the bacteria would absorb the glo DNA since it has ampicillin and Isopropyl β-D-1-thiogalactopyranoside so those would help it transform. Another plate that would have genetically transformed bacteria would be +DNA/+AMP. Since the DNA plasmid placed with this bacteria, the bacteria will have transformed to resist the ampicillin's antimicrobial properties.
3. What is the purpose of the control plates? Explain the difference between the
controls and why each one is necessary.
The purpose of the control plates is to test different factors that may affect the bacteria. They'll also tell you how certain things may change the transformation of the bacteria. The Regular -DNA plate will have the original bacteria that will help you compare the +DNA plates to. The -DNA/+AMP will then tell you if the +DNA/+AMP has transformed or not based on if they have actually grown.
4. Why would one compare the -DNA/+Amp and +DNA/+Amp plates?
You would compare the -DNA/+AMP and +DNA/+AMP plates to see if one was more affected by the antibiotic than the other. Since the +DNA has plasmid they should be resistant to the ampicillin. You'd want to see how the plates react differently from each other. They are the same bacteria but +DNA has been transformed and you want to make certain of it.
Protocols:
Hypothesis:
If the bacteria survive once the P-glo is added and then glow once in the IPTG plate then it has successfully been transformed.
Data:
*Data used is based off of Meghana's and Stephany's bacterial plates*
Bacteria can transfer DNA by using either transformation or conjugation. Transformation and conjugation are different in the aspect of how the bacteria receives the DNA. In transformation the bacterium grabs any extra DNA from its environment while in conjugation one bacteria inserts its DNA into another bacteria.
In this lab we will be transforming E.Coli bacteria with ampicillin resistant and glowing plasmids. You will have 2 +DNA plates. One with AMP to see if they have been successfully transformed and a plate containing IPTG which will help induce the lac operon which will make the transformed bacteria glow under U.V. light.
*anyone allergic to penicillin, ampicillin, kanamycin, or tetracycline should not participate*
Agar plates should already be prepared. Aseptic techniques must be used and proper PPE must be worn.
Materials:
- 2 microcentrifuge tubes
- E.Coli
- 4 Agar plates
- Ampicillin
- toothpick
- inoculation loops
- 6 pipets (just in case)
- P-glo plasmid
- IPTG
- ice bath (shared)
- Incubators (2)
- U.V. light
Pre-lab questions:
1. On which plate(s) would you expect to find bacteria most like the E. coli on the
source plate? Explain.
I would probably expect to find bacteria like the E.Coli on the source plate -DNA. I would expect that one since no plasmid would have been added and there are no antibiotics being applied; it would be the same bacteria from the beginning.
2. On which plate(s) would you find only genetically transformed bacterial cells?
Why?
You would find strictly genetically transformed bacterial cells on the +DNA/+AMP/+IPTG because the bacteria would absorb the glo DNA since it has ampicillin and Isopropyl β-D-1-thiogalactopyranoside so those would help it transform. Another plate that would have genetically transformed bacteria would be +DNA/+AMP. Since the DNA plasmid placed with this bacteria, the bacteria will have transformed to resist the ampicillin's antimicrobial properties.
3. What is the purpose of the control plates? Explain the difference between the
controls and why each one is necessary.
The purpose of the control plates is to test different factors that may affect the bacteria. They'll also tell you how certain things may change the transformation of the bacteria. The Regular -DNA plate will have the original bacteria that will help you compare the +DNA plates to. The -DNA/+AMP will then tell you if the +DNA/+AMP has transformed or not based on if they have actually grown.
4. Why would one compare the -DNA/+Amp and +DNA/+Amp plates?
You would compare the -DNA/+AMP and +DNA/+AMP plates to see if one was more affected by the antibiotic than the other. Since the +DNA has plasmid they should be resistant to the ampicillin. You'd want to see how the plates react differently from each other. They are the same bacteria but +DNA has been transformed and you want to make certain of it.
Protocols:
- Label one microcentrifuge tube with “+DNA” and the other with “-DNA”
- Transfer 50 microliters ice-cold CaCl2 into the -DNA tube using a sterile 1 mL pipet
- Using a toothpick transfer 15 well-isolated colonies from the E.Coli plate to the -DNA tube
- Twist the toothpick between your fingers to free the cells. Resuspend the bacterial cell in the CaCl2 by swirling vigorously until no clumps are visible and the cell suspension looks cloudy
- Transfer 250 microliters of the cell suspension to the tube labeled +DNA and place the tubes on ice
- Add 10 microliters of pFluoroGreen DNA to the tube labeled +DNA and DO NOT add it to the -DNA tube
- Incubate the tubes for 10 minutes on ice
- Place tubes in a 42 C water bath for 90 seconds
- Return to ice bucket for 2 minutes
- Transfer 250 Microliters of recovery broth to each tube using a sterile 1 mL pipet and gently mix by flicking the tube
- Incubate for 30 minutes in 37 C water bath
- Label the bottom of four agar plates as indicated:
- -DNA (no stripe)
- -DNA/+AMP (one stripe)
- +DNA/+AMP (one stripe)
- +DNA/+AMP/+IPTG (two stripes)
- After recovery, remove the tubes from the water bath and place them on lab bench
- Using sterile 1 mL pipet, transfer 250 microliter recovered cell from -DNA to the middle of the -DNA and -DNA/+AMP plates
- Transfer 250 microliters of +DNA to the middle of the +DNA/+AMP and the +DNA/+AMP/+IPTG plates
- Spread the cells over the plate. Sterilize or change the loop before spreading the the next sample and then cover the plates and wait for five minutes for the cell suspension to be absorbed by the agar
- Seal the plates with parafilm, label and incubate in a 37 C incubator for 16-18 hours. If no incubator is present leave at room temp for 24-48 hours
- Observe the plates using a long wave U.V. light and then record the number of colonies on the plate and the color of the bacteria under the UV light
Hypothesis:
If the bacteria survive once the P-glo is added and then glow once in the IPTG plate then it has successfully been transformed.
Data:
*Data used is based off of Meghana's and Stephany's bacterial plates*
Plates |
Growth |
Color |
Description |
Efficiency |
-DNA (1) |
lawn |
Cloudy brown creme color but does not glow |
Bacterial lawn has grown in the plate. The lawn is a tannish brown. Most people’s plates look like this because it was the original bacteria plated. |
- |
-DNA/+AMP (2) |
no growth (0) |
no color, plain agar and does not glow |
Nothing is on the plate because nothing should be on this plate. The ampicillin has killed off all of the non-transformed E.Coli leaving the plate as just agar. |
- |
+DNA/+AMP (3) |
400 |
Plain E.Coli. Colonies are cloudy creme color, does not glow. |
3 clumps of colonies have grown on the plate meaning that the bacteria have been transformed. Unlike the bacterial lawn they are a creme color. Most of the class got similar outcomes. |
(400/1000 micrograms)*(0.5 ml/0.25 ml)= 0.08 transformants per micrograms |
+DNA/+AMP/+IPTG (4) |
600 5 dense colonies glowing |
Glows under U.V. as greenish blue. |
5 major colonies have grown; glowing. There are 2 clumps of colonies and a giant heap of about 250 colonies. Everyone’s plates are different due to the fact that they cut the time for incubation short and/or didn’t let the plates sit and soak for long enough. |
(600/10000 micrograms)*(0.5 ml/0.25 ml)= 0.12 transformants per micrograms |
Actual photos!:
Post-Lab questions:
1. Exogenous DNA does not passively enter E. coli cells that are not competent. What treatment do cells require to be competent?
Cells have to undergo cell suspension. When they go through this process the reaction takes place and it catalyzes the reaction so the bacteria can take in the extra DNA and transform. Cell suspension allows the bacteria to take in the DNA and insert it with the rest of its own DNA.
2. Why doesn’t the recovery broth used in this experiment contain ampicillin?
The recovery broth used in the experiment doesn't contain ampicillin, an antibiotic, because it is applied to both the -DNA and the +DNA tubes and if the ampicillin was added to the -DNA tube it would kill the bacteria that would be plated later. It would leave no control bacteria for the other plates to be compared to.
3. What evidence do you have that transformation was successful?
The Bacteria was transformed successfully based on the evidence the ampicillin plates give us. The bacteria without the the DNA plasmid dies because it doesn't have the genes to survive but the bacteria that contains the bacteria survives the ampicillin.
4. What are some reasons why transformation may not be successful?
Transformation may not be successful because the bacteria may not have been suspended long enough or the bacteria wasn't even able to survive.
5. What is the source of the fluorescence? Why are some cells fluorescent and other cells not fluorescent?
The source of the fluorescence is the plasmid mixed with the bacteria. Some cells (+DNA/+AMP) are not fluorescent because they weren't introduced to the IPTG which helps them metabolise. When the cells are exposed to the IPTG and when they do metabolise the plasmid they are able to induce the lac operon and glow.
Conclusion:
In the end, based off of the hypothesis, our bacteria were transformed (the ones used for data). Our classmates bacteria grew beautifully and fully.
Bacteria have to take in a piece of random DNA in their environment in order to transform without another bacteria. Once they transform these E.Coli bacteria were able survive in ampicillin, a substance that normally kills E.Coli. The transformed bacteria, when introduced to IPTG, are then able to glow because the glow gene is shown when the cells metabolize.
In my group's experiment our +DNA plates did not grow much bacteria because of something we messed up when we were preparing to plate our bacteria. We had 2 plastic inoculation loops; the one for +DNA plates was dropped and had to be disinfected in order to be used so we thought it be best that we spray it with alcohol. When we did this we didn't know exactly when the alcohol had dried so we spread our bacteria and the alcohol most likely killed the bacteria for the +DNA plates.
1. Exogenous DNA does not passively enter E. coli cells that are not competent. What treatment do cells require to be competent?
Cells have to undergo cell suspension. When they go through this process the reaction takes place and it catalyzes the reaction so the bacteria can take in the extra DNA and transform. Cell suspension allows the bacteria to take in the DNA and insert it with the rest of its own DNA.
2. Why doesn’t the recovery broth used in this experiment contain ampicillin?
The recovery broth used in the experiment doesn't contain ampicillin, an antibiotic, because it is applied to both the -DNA and the +DNA tubes and if the ampicillin was added to the -DNA tube it would kill the bacteria that would be plated later. It would leave no control bacteria for the other plates to be compared to.
3. What evidence do you have that transformation was successful?
The Bacteria was transformed successfully based on the evidence the ampicillin plates give us. The bacteria without the the DNA plasmid dies because it doesn't have the genes to survive but the bacteria that contains the bacteria survives the ampicillin.
4. What are some reasons why transformation may not be successful?
Transformation may not be successful because the bacteria may not have been suspended long enough or the bacteria wasn't even able to survive.
5. What is the source of the fluorescence? Why are some cells fluorescent and other cells not fluorescent?
The source of the fluorescence is the plasmid mixed with the bacteria. Some cells (+DNA/+AMP) are not fluorescent because they weren't introduced to the IPTG which helps them metabolise. When the cells are exposed to the IPTG and when they do metabolise the plasmid they are able to induce the lac operon and glow.
Conclusion:
In the end, based off of the hypothesis, our bacteria were transformed (the ones used for data). Our classmates bacteria grew beautifully and fully.
Bacteria have to take in a piece of random DNA in their environment in order to transform without another bacteria. Once they transform these E.Coli bacteria were able survive in ampicillin, a substance that normally kills E.Coli. The transformed bacteria, when introduced to IPTG, are then able to glow because the glow gene is shown when the cells metabolize.
In my group's experiment our +DNA plates did not grow much bacteria because of something we messed up when we were preparing to plate our bacteria. We had 2 plastic inoculation loops; the one for +DNA plates was dropped and had to be disinfected in order to be used so we thought it be best that we spray it with alcohol. When we did this we didn't know exactly when the alcohol had dried so we spread our bacteria and the alcohol most likely killed the bacteria for the +DNA plates.