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Biotechnology: Bacterial Transformation Lab

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Ezra Hall

on 9 December 2013

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Transcript of Biotechnology: Bacterial Transformation Lab

We expected to see bacterial growth on the LB/amp pGLO positive plate because the bacteria should have absorbed the plasmid that was resistant to ampicillin. We also expected the LB/amp/ara pGLO positive plate to have glowing colonies because it had arabose sugar that should have activated it. Neither of these plates had growth on them and can be attributed to errors in our process. We believed that we did not "heat shock" the cells properly resulting in the lack of growth.
- Reece, Jane B., and Neil A. Campbell. "Genetics - Biotechnology". Campbell Biology/Jane B. Reece... [et al.]. San Francisco, CA: Benjamin Cummings, 2011. 398. Print.
- Anderson, Mr. "AP Biology Lab 6: Molecular Biology" YouTube. YouTube, 10 May 2013. Web.
- AP Biology Investigative Labs Manual (College Board, 2012), Investigation 8 - Biotechnology: Bacterial Transformation
Prokaryotes are singled celled organisms and are the best understood microorganisms. The purpose of our experiment was to show how to transform DNA in a way that would make it express new characteristics. In the experiment we will be using inoculation loops, Laurel Broth, and transformation solution as well as heat shock and incubation to insert pGLO DNA into an E.coli bacteria. The outcome of this experiment should be that the petri dish with pGLO and arabanose sugars will have glowing bacteria colonies.The only one of our plates that had growth on it was the LB pGLO- one. This leads us to believe that the pGLO plasmid did not combine with the E.coli DNA. Our plate with ampicillin also did not have growth which was expected
Biotechnology: Bacterial Transformation Lab
Our experiment yielded results that were unexpected. One thing that we noticed was that the pGLO pates were the ones that did not have expected results. Both the LB/amp/ara pGLO + and the LB/amo pGLO + did not have any growth. The ampicillin resistant gene in the pGLO should have created bacteria growth but ultimately did not leading us to believe that there was an error in our process.
The purpose of the lab was to learn how transformation occurs and to see the results and consequences of transformation. Our experiment yielded some unexpected results as we discovered no growth on any of the plates except for the LB pGLO - plate which had approximately 320 small bacterial colonies growing on it. These results did not support scientific theories and did not follow the expected outcomes. One thing that we were able to prove with our experiment was that if plasmids and bacteria are not combined in the right way then bacterial DNA will not be able to take up the plasmid. There are a number of things that we could have done to prevent the errors that occurred in our lab. The main error in our lab was that the pGLO + plates did not have any growth. This was probably due to improper "heat shock" and an error in the ice bath or water bath procedures. We could have made sure that our micro tubes were properly immersed in both ice baths for the proper periods of time. To ensure this we could have inserted the foam floater into the ice bath to make sure the were properly submerged in the ice. Another error could have been that we did not gather enough bacteria to put into the solution. We also could have had in error in the spreading of bacteria on the plates which if improved could have yielded better results.
Bacterial transformation occurs when bacterium absorbs a naked DNA located on there surface and integrates it into there own DNA. The incorporation of this naked DNA usually leads to the expression of new traits in the bacterium. Plasmids are usually used in bacterial transformation because they are small circular DNA molecules that are easily incorporated in the host bacterium's DNA. In our lab we used E.Coli bacteria that is going to be transformed to incorporate a pGLO plasmid with ampicillin resistance, an arabanose sugar and Glowing Fluorescent Proteins. Ampicillin is an antibiotic that would usually kill the cell but this is prevented by the gene on the pGLO with resistance to it. The Glowing Fluorescent Proteins should cause the bacteria to glow if its RNA is able to be transcribed and translated. The arabanose sugar helps the GFP's become transcribed because it stops the repressor from binding to the operon on the GFP gene. If the repressor were attached it would prevent RNA polymerase from transcribing the gene and stopping the production of GFP's.

The first step in the lab is to acquire two micro tubes, one for the - plasmid and one for the + plasmid. The next step is to use a pipette to add 250 micro liters of CaCl2 or transformation fluid into each tube. Then take a sterile inoculation loop and acquire a colony of E.coli bacteria from the starter plate. Add the bacteria to one of the micro tubes by submersing it in the transformation fluid and by spinning the loop. Then repeat this step but add the bacteria to the other tube. Next pipette 10 micro liters of plasmid solution into the + plasmid micro tube. Then make sure both of the tubes are sealed tight and incubate them in ice for 10 minutes (make sure the tops of the tubes are not submerged in the ice). While you are waiting you can label your agar plates (LB/AMP - pGLO, LB, LB/amp +pGLO, LB/amp/ara). When the tubes are done in the ice put them in a test tube float and insert it into a water bath set at 42 degrees Celsius for only 50 seconds. Next put te tubes back on ice for two minutes. Then pipette 250 micro liters of LB nutrient broth to each tube. Next pipette 100 micro liters of each solution onto the according plates. Then use four sterile inoculation loops to spread the solutions on the agar plates. Finally put the plates in an incubator set at 37 degrees Celsius for 24 hours, make sure the plates are upside down to prevent condensation.
Purpose Statement
The purpose of the lab was to learn how transformation occurs and how to do a transformation lab. We also learned the importance of transformation in cells as well as the results and consequences that occur due to transformation
# of colonies observed
LB/amp (pGLO -) 0
LB (pGLO -) 327
LB/amo (pGLO +) 0
LB/amp/ara (pGLO +) 0
LB (pGLO negative)
a large number of white convex colonies were observed on the plate
LB/amp pGLO negative

There was no growth observed on this plate due to the presence of ampicillin, which killed all of the bacterial growth. Ampicillin breaks down the cell membrane which ultimately kills the cell.

LB pGLO negative

This was one of our control plates and was the only plate on which we found substantial bacterial growth. On this plate we found approximate 320 bacterial cultures. One thing that was noticed on this plate was that the bacteria only grew on one side of the plate when it should have been covered like a lawn. The best explanation for this is that the bacteria was not properly spread on the plate using the inoculation loop.
LB/amp (pGLO +) and LB/amp/ara (pGLO +)

On the two pGLO positive plates there was no observed growth of bacteria. This went against the expected results as we should have seen growth on both plates and the LB/amp/ara plate should have had glowing colonies. The pGLO DNA if properly incorporated should have resisted the ampicillin leading to growth. This leads us to believe that the pGLO DNA was not properly taken up by the E.coli and we must have had an error somewhere in our lab.
Due to the results we must reject our alternate hypothesis and accept out null hypothesis. The pGLO had not bacterial growth and the LB/amp/ara pGLO + plate did not glow.
Alternate Hypothesis
Our alternate hypothesis for the experiment is that that pGLO DNA will incorporate in the E.Coli DNA and produce new traits. The LB/amp (pGLO -) will be the only plate with no growth. The LB (pGLO-) plate will have a lawn of growth while the LB/amp (pGLO +) and the LB/amp/ara (pGLO +) plates should have a number of colonies with the plate with ara having glowing colonies
Null Hypothesis
Our null hypothesis for the experiment is that the pGLO DNA will not properly incorporate into the E.Coli DNA. If the pGLO is not taken up by the E.Coli DNA then the bacteria will be killed by the ampicillin and there will be no growth in the LB/amp pGLO-, LB/amp pGLO+ and the LB/amp/ara pGLO+ plates. The LB pGLO- plate will still have growth across the surface.
We predict that the alternate hypothesis will be correct.
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