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Genetic Transformation of E.Coli

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Emily Kiely-Smith

on 10 December 2013

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Transcript of Genetic Transformation of E.Coli

Genetic Transformation of E.Coli
Making E.Coli Glow
Abtsract
Introduction
Methods
Results
Discussion
Conclusion
References
THANKS FOR WATCHING!
Presentation by: Emily Kiely-Smith
Materials
Individual Workstation
-E. coli starter plate
-4 poured agar plates as follows:
-1 LB agar plate
-2 LB/amp agar (LB agar containing ampicillin) plates
-1 LB/amp/ara agar (LB agar containing arabinose sugar) plate
-Transformation solution (CaCl2, pH 6.1) kept ice cold
-LB nutrient broth
-Sterile innoculation loops
-100-1000 µL sterile bulb pipettes
-1-10 µL micropipettes with sterile tips
-Microcentrifuge tubes
-Microcentrifuge tube holder/float
-Container full of crushed ice
-Marking pen
General Workstation:
-DNA plasmid (pGLOat 0.005 µg/µL)
-42°C water bath and thermometer
-37°C incubator
-20 μL adjustable-volume micropipettes
and tips (optional)
-10% household bleach
-Biohazardous waste disposal bags
-Masking or lab tape
Procedure
1. Familiarize yourself with sterile technique, materials and lab equipment, and safety procedures for handling bacteria and decontaminating the work area.
2. Label one closed microcentrifuge tube (micro test tube) “+ pGLO” (+ means it
will have
the plasmid) and one tube “-pGLO” (- means it
will not have
the plasmid). Place them in the microcentrifuge tube holder/float.
3. Carefully open the tubes and, using a 100–1000 μL bulb pipette with a sterile tip, transfer 250 μL of the ice cold transformation solution (CaCl2) into each tube.
4. Place both tubes into the ice.
5. Use an inoculation loop to pick up a single colony of bacteria from your starter plate. Be careful not to scrape off any agar from the plate. Pick up the “+ pGLO” tube and immerse the loop into the transforming solution (CaCl2) at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the solution. Appropriately discard the loop.
6. Use a new sterile 100–1,000 μL micropipette to repeatedly pulse the cells in solution to thoroughly resuspend the cells. (Note that the clear transformation solution will become cloudy as the E. coli cells are suspended.) Place the tube back on the ice.
7. Using a new inoculation loop, repeat steps 5 and 6 for the “- pGLO” tube, being careful to keep your nose and mouth away from the tip end when pipetting.
8. Using a 1–10 μL micropipette with a sterile tip, transfer 10 μL of the plasmid solution directly into the E. coli suspension in the “+ pGLO” tube. Tap tube with a finger to mix, but avoid making bubbles or splashing the suspension up the sides of the tube. Do not add the plasmid solution into the “- pGLO” tube.
Introduction, Continued
Background
Safety Procedures
and Correct Lab Techniques
Hypotheses and Prediction
-Null Hypothesis: the pGLO plasmid will not affect the physical or genetic characteristics of the E. coli bacteria. (Any phenotypic change will not be the result of the pGLO)

-Alternative Hypothesis: the pGLO plasmid will affect the physical and genetic characteristics of the E. coli bacteria. (Any phenotypic change will be the result of the pGLO)

Based on previous scientific evidence, I predict that the pGLO plasmid will have an effect on the physical and genetic characteristics of the the E. Coli bacteria.

Video on Plasmids and Restriction Enzymes
Video Explaining the Experiment (watch until 5:09)
Deoxyribonucleic Acid (DNA) is the molecule that contains our genetic information. DNA is made up of nucleotides, which are the "building block(s) of a nucliec acid"( Textbook G-24). Specific sequences of nucleotides, also known as genes, code for specific information. Genes get passed down from generation to generation, and therefore so does the information they carry. One gene that is relevant to our experiment naturally occurs in deep sea creatures; a bioluminscence gene that allows creatures to "glow in the dark". In our experiment, this gene has been taken from a jelly fish and inserted into a plasmid. Plasmids are small sections of bacteria that can be passed between cells to provide genetic variation. For a more detailed explanation of plasmids, watch the video on the left entitled "video on plasmids". In our lab, humans have engineered a plasmid to contain a bioluminesence gene and also a gene for antiobiotic (in this case ampicillin) resistance. If the E. coli bacteria takes up the plasmid, it should, in theory, be able to grow in ampicillin. So, 3 of the 4 plates for growing bacteria will contain ampicillin, to test the effectiveness of the pGLO plasmid for antibiotic resistance.
Procedure Continued
9. Incubate both tubes (“+ pGLO and “- pGLO”) on ice for 10 minutes. Make sure the bottom of the tubes make contact with the ice.
10. While the tubes are sitting on ice, label each of your agar plates on the bottom (not the lid) as "+" or "-" for pGlO.
11. Following the 10-minute incubation at 0°C, remove the tubes from the ice and “heat shock” the cells in the tubes. It is critical that the shock is received. Make sure the tubes are closed tightly! Place the tubes into a test tube holder/float, and dunk the tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the holder so that the bottom of the tubes with the suspension makes contact with the water.
12. When the 50 seconds have passed, place both tubes back on ice. For best transformation results, the change from 0°C to 42°C and then back to 0°C must be rapid. Incubate the tubes on ice for an additional two minutes.
13. Remove the holder containing the tubes from the ice and place on the lab counter. Using a 100–1,000 μL micropipette with sterile tip, transfer 250 μL of LB nutrient broth to the “+ plasmid” tube. Close the tube and gently tap with your finger to mix. Repeat with a new sterile micropipette for the “- plasmid” tube.
14. Incubate each tube for 10 minutes at room temperature.
15. Use a 10–1,000 μL micropipette with sterile tip to transfer 100 μL of the transformation (“+ pGLO”) and control (“- pGLO”) suspensions onto the appropriate LB and LB/Amp plates. Be sure to use a separate pipette for each of the four transfers.
16. Using a new sterile inoculation loop for each plate, spread the suspensions evenly around the surface of the agar by quickly “skating” the flat surface of the sterile loop back and forth across the plate surface (Figure 3). Do not poke or make gashes in the agar! You might also use small sterile glass beads to spread the suspensions by gently rocking the beads across the surface of the agar. Allow the plates to set for 10 minutes.
17.Stack your plates and tape them together. Place the stack upside down in the 37°C incubator for 24 hours.
Observations
LB, "-pGLO": showed a "lawn" of bacteria
LB/amp "-pGLO": showed no growth
LB/amp "+pGLO": showed growth. no bioluminesence observed
LB/amp/ara "+pGLO": showed growth and bioluminesence








Calculations
Transformation Efficiency= Total number of colonies growing on the agar plate
Divided by: Amount of DNA spread on the agar plate (in μg)

Transformation Efficiency (T.E.) = 39 (colonies)/ 0.8 μg = 48.75

Expected T.E.= 8.0e2

Perecent Error = | 48.75-8.0e2| / (8.0e2) x 100 = 93.9% error

Purpose
In this lab we hope to determine the effect of the pGLO plasmid on E. coli bacteria.
The Theory Behind Bacterial Transformation
In the introduction, the basics of bacterial transformation were discussed, as were the expected outcomes of the experiment. We expected to observe the following:
A "lawn" of bacteria in the LB plate "-pGLO"
NO growth in the LB/amp plate "-pGLO"
Growth, but no bioluminescence in the LB/amp plate "+pGLO"
Growth and bioluminescence in the LB/amp/ara plate "+pGLO"

This is based on the extensive research done in the field of biotechnology*, and plasmids and their effects on bacteria are now a part of the grade 12 biology curriculum (in Manitoba). These outcomes are the product of scientific facts, based on previous theory and research. It is known how plasmids work as shown in Campbell Biology (297) and in other sources, such as the youtube videos I have posted.
But what is the reasoning behind each expected outcome?

*The manipulation of organisms or their components to produce useful products. (Campbell Biology, G-4)

It is important to understand proper safety procedures before starting your lab. Always remember to:
-wear gloves and aprons, keeping long hair tied up
-wash down all surfaces with bleach before AND after the experiment
-avoid breathing when the E. coli bacteria is exposed
-use proper lab techniques (as shown in the video to the right)
Follow these steps to avoid any accidents and ensure the lab runs smoothly.
The experiment, explained in full by the video to the right, has expected outcomes based on previous scientific trials and theories. We will expect that any bacteria cell that takes up the plasmid will exhibit both antibiotic resistance and bioluminescence, under the right conditions. Each plate will contain Luria Broth (LB) agar, a nutrient-rich medium required for bacterial growth. Three plates will also contain ampicillin. It is expected that normal bacteria will die when grown in ampicillin (because it is an antibiotic which kills bacteria), so one plate will contain normal bacteria, aka no plasmid, and ampicillin(amp) as a control. Bacteria that have taken up the plasmid should live (due to the antibiotic resistance gene). So, two of the plates will contain bacteria with a plasmid and ampicillin(amp), to demonstrate that the plasmid effects antiobiotic resistance. One of these will also contain the sugar arabinose(ara), which is needed for the cells to become bioluminescent. Based on scientific theory, the LB plate will exhibit high bacterial growth (a bacterial "lawn"). The plate with LB and amp grown with normal bacteria will exhibit no growth. The plate with bacteria positive for the plasmid, grown in LB and amp, but without arabinose, should show growth, but no bioluminescence. The final plate, containing LB, amp, ara and bacteria with the plasmid, should show both growth and bioluminscence. It is important you know this beforehand and start actively thinking about the outcomes BEFORE you do the experiment,
- AP® Biology Investigative Labs: An Inquiry-Based Approach. New
York:
The College Board 2012. Web. 8 Dec. 2013.

-Reece, Jane B. Lisa A. Urry. et al.
AP* Edition Campbell Biology.
San Fransisco: Pearson Benjammin Cummings, 2011. Print.
Rejected or Accepted?
-Null Hypothesis: the pGLO plasmid will not affect the physical or genetic characteristics of the E. coli bacteria. (Any phenotypic changes will not be a result of the pGLO)

-Alternative Hypothesis: the pGLO plasmid will affect the physical and genetic characteristics of the E. coli bacteria. (Any phenotypic changes will be a result of the pGLO)
To reject or accept either hypothesis, we must compare the two plates that had the exact same conditions, but with the different types of bacteria (one with the plasmid, and one without). The bacteria without the plasmid in the LB/amp plate ALL died. Some of the bacteria with the plasmid in the LB/amp plate survived. It can be concluded from this evidence that it was the plasmid that transformed the bacteria to be able to resist ampicillin (since that was the only factor that was different between the two). Therefore, the plasmid caused a genetic, and also physical, change to the E. coli. So we will reject the null hypothesis and accept the alternative hypothesis.

I also made a prediction that the alternative hypothesis would be accepted, based on the thorough research done in this subject. This means my prediction was correct.
Reasons For Expected Outcomes
Error
It is expected that normal bacteria will die when grown in ampicillin (because it is an antibiotic which kills bacteria), so one plate will contain normal bacteria, aka no plasmid, and ampicillin(amp) as a control. Bacteria that have taken up the plasmid should live (due to the antibiotic resistance gene). So, two of the plates will contain bacteria with a plasmid and ampicillin(amp), to demonstrate that the plasmid effects antiobiotic resistance (and should show growth). One of these will also contain the sugar arabinose(ara), which is needed for the cells to become bioluminescent. The LB plate will exhibit high bacterial growth (a bacterial "lawn") since the LB medium is nutrient rich (and bacteria need nutrients to survive. The plate with LB and amp grown with normal bacteria will exhibit no growth(because the ampicillin will kill it). The plate with bacteria positive for the plasmid, grown in LB and amp, but without arabinose, should show growth (because it has developed antibiotic resistance from the plasmid), but no bioluminescence (because arabinose is not present). The final plate, containing LB, amp, ara and bacteria with the plasmid, should show both growth and bioluminscence (because all elements are present).
*Copied From Introduction With Slight Variation, Skip This Slide If No Recap Is Needed*
We found that the expected phenotypic outcomes for the experiment matched our observed outcomes. We found growth and bioluminescence on the appropriate plates. We also calculated the transformation efficiency, which was extremely different from the expected transformation efficiency. Our transformation efficiency was 48.75, while the expected efficiency was 8.0e2 (800). This meant that our percent error was 93.9%, a staggering amount to have in a lab. This may have been due to the fact that, while transferring the bacteria, we touched the innoculation loop to the bottom and sides of the micro tubes, with quite some force. This may account for some of the error, since we might have killed some of the bacteria by doing that. Other errors might have occurred due to a sample contamination, inproper timing during the procedure, or other such careless mistakes. Finally, some error was introduced due to the equipment, which is not as "high tech" as it could have been.
We successfully determined the effect of the pGLO plasmid on the E. coli bacteria. It was determined that pGLO gives bacteria both antibiotic resistance to ampicillin, and also bioluminescence in the presence of arabinose. Although the expected results matched our experimental ones, the bacteria was not transformed as effectivley as was expected. In fact, our error was 93.9% for that part of the lab, due to many possible sources of error. These results relate directly to biotechnology, as plasmids are extremely useful tools to ensure a bacterial gene is expressed. They are often used in the production of certain hormones, such as insulin, which can be live-saving to those with diabetes. Perhaps if our group took more care in the transfer of bacteria, and timing the steps, our results might have had less error, and therefore be more noteworthy. Perhaps if we were to repeat the experiment again, we could research the effects of different variables on the transformation efficiency of the plasmid; this could be greatly benefitial to biotechnology to know which in conditions are best for bacterial transformations. Overall, the lab itself was still successful and noteworthy in it's own way.
This bacterial transformation lab examines the actions of the human-engineered pGLO plasmid in relation to E. coli bacteria. In our experiment, we hoped to determine the plasmid's effect on the E. coli bacteria. Bacteria was transferred from a pre-grown starter plate with an innoculation loop, and put in a micro test tube. One micro test tube contained bacteria with the plasmid, and one contained bacteria without. There were three different growth media used; Luria Broth(LB), LB/ampicillin(amp), and LB/amp/arabinose sugar (ara). The plasmid was added to a plate with LB/amp and LB/amp/ara. The plasmid wasn't added to the plate with LB, or to a second plate with LB/amp. Our data showed that the LB plate (without pGLO) experienced a bacterial "lawn" of growth, as expected for a bacteria grown in normal conditions. The LB/amp plate (without pGLO) showed no growth at all. However, the LB/amp plate WITH pGLO showed some growth. From this, it can be concluded that pGLO, somehow, causes a change that creates antibiotic resistance. Based on previous scientific research, it is known that plasmids can transform the genes of bacteria, so it can be assumed that this change caused the antibiotic resistance. The final plate was grown with LB/amp/arabinose sugar (a sugar that is needed to create bioluminescence), and experienced growth and bioluminescence, where as no other slide showed bioluminescence. This offered conclusive proof that the pGLO plasmid affected E. coli bacteria by giving the bacteria both bioluminscent and antibiotic resistant properties.
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