Pvib Bacterial Transformation Lab

pVIB Bacterial Transformation Lab By Rebecca Varghese AP Biology Per. 1-2 ABD Mr. Resch November 21st, 2008 Abstract: Genetic transformation is changing the genetic material of an organism by inserting different genes with the purpose of changing an organism’s trait/s. In order to alter the genetic make-up of

E. coli, it was necessary to move genes from one organism to another with the help of plasmids.

The plasmid for luminescence, the Lux operon, contained an ampicillin resistance gene. Using calcium chloride, we transferred isolated colonies of E. coli to a tube filled with plasmid DNA

(one without as the control). After heat shock, Luria broth was added to each tube. Both the control and the experimental tube contents were spread onto the plasmid plates that provided a suitable environment for bacteria to reproduce in. After about 36 hours, the plate that had ampicillin glowed, but the positive plasmid plate, the negative plasmid plate, and the plate without ampicillin did not glow. We believe this is because luciferin will only cause E. coli to glow in the presence of ampicillin, which triggers transcription of the gene in the plasmid.

Introduction: The purpose of this experiment was to determine how genetic transformation using the lux operon in E. coli would affect its ability to glow. Genetic transformation is a common type of genetic manipulation in which one gene, or a piece of DNA, is inserted into another’s to code for a specific trait (Redfield). This process is used in a wide range of fields, from agriculture to medicine. This has provided the public with many advantageous products and possible cures in the medical field that have been achieved through insertion of different genetic strains.

Ampicillin is an antibiotic that E.Coli is naturally not resistant to, but the plasmid (a loose segment of DNA) that we transferred contained the lux operon and an ampicillin resistant gene.

In E. coli, luminescence is caused by the gene luciferase, an enzyme that emits light during its catalysis. Luciferase is termed “heterodimer”, and is made up of two polypeptides made up of alpha and beta subunits (the active site is found in the alpha subunit) (Watts). The binding patterns between the alpha and beta subunits during catalysis causes the alpha subunit to change its conformation (Campbell). This gene is found in the lux operon, and this operon contains 5 different genes that are all involved in the process of emitting visible light, in addition to two more that regulate the lux operon. Luciferins are pigments that are physically responsible for luminescence. They are small molecule-substrates that bind to luciferase and create energy in the form of light. The recombination of genetic material will force the E.coli strain being used to contain this combination of the lux operon, luciferase, and luciferins (all interconnected), and will either cause the bacteria to glow or to remain non-luminescent. With the use of calcium chloride and heat shock, the DNA will be able to enter the cell and help the bacteria absorb it, and eventually contain the desired plasmid DNA (Redfield). Calcium chloride is used in the test tube solution of the bacteria and plasmid so that the negative charges in the bacteria and the DNA phosphate backbone both neutralize and the charges do not repel each other (Roe). Heat shock therapy is a drastic change in temperature for the test tubes filled with colonies of E.coli, and this will induce the plasmid to open up and combine genetic material (Roe). Because of this genetic switch, we predicted that the agar plates (used to encourage bacterial growth and reproduction) with ampicillin and transformed bacteria would glow in the dark. The agar plates without ampicillin, and transformed bacteria would grow on the ampicillin plate, but both the untransformed bacteria and the plates without ampicillin would remain non-luminescent. Results Table:

+ PLASMIDS -PLASMIDS +AMP -AMP

One large, thinly No signs of bacterial Smaller clumps of No signs of bacterial spread smear of growth white growth cloudy layer No change Colonies are about No change Cloudy white in Agar plate remains the the size of a pinhead Agar plate remains color same color- white/yellow 14 colonies the same color- No glow Colony size varies white/yellow More abundant from point to point No glow strains around Strong glow edges No glow

Discussion Questions:

Please describe, at the cellular/molecular level, the precise steps involved in heat shock. That is, how can we force a bacterial cell to take up a plasmid?

Heat shock is used after a series of steps. The addition of Calcium Chloride stops the DNA and bacterial cell wall from repelling each other by neutralizing their charges. After a 15-minute waiting period, the solution containing the plasmid DNA, bacteria, and Calcium Chloride slow the molecules of DNA and molecules in the phospholipid bilayer. Because the cell wall (containing the phospholipid bilayer) had constant movement, the reduction in movement leaves gaps between the cell wall. Heat shock is used to stimulate DNA to pass through that cell wall, because the addition of heat raises the kinetic energy that exists between molecules. The plasmids then are induced to enter the cell and the bacterial cell can accept the plasmid.

If any of the predictions regarding bacterial growth made in the pre-lab considerations differed from your observed results, please describe them and explain why you believe you obtained these results.

We predicted that the agar plate without ampicillin (but with plasmid DNA) would glow in the dark.

However, there were no luminescent bacteria present in the plate. This can be for several reasons. The amount of luminescent DNA might not have been enough to fully glow in our circumstances, but in total darkness and with the aid of microscope- there may or may not be traces of luminescent DNA. The bacteria that grew and reproduced substantially were those that contained the lux operon, and luminescent clusters were visible in 14 isolate colonies on our agar plates. The other explanation that can be offered is that DNA is only luminescent in the presence of ampicillin- and this triggers transcription of the gene in the plasmid.

Using the logic, DNA cannot be ampicillin resistant and still be luminescent, and E.coli strains are naturally not resistant to ampicillin unless they contain the lux operon (which contains an ampicillin resistant gene) and this will result in non-luminescent DNA.

What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have taken up the plasmid)?

In this experiment, the ultimate goal was to see how much plasmid DNA had been accepted by the bacterial cell- and this would measure the success of the genetic transformation. Because the plasmid contained the lux operon, which is naturally ampicillin resistant, both of these traits could be used to measure the growth of bacteria (the existence of either one of these traits). Luminescence is easily tested for, and if the bacterial cells glow in the dark, it will be easy to tell if the E. coli cells accepted the plasmids. The use of controls was effective as well, but if the plate that was positive for ampicillin still contained transformed bacteria, then the bacteria that had contained the ampicillin resistant gene would have been shown to be accepted by the bacterial cell.

Transformation efficiency is expressed as the number of antibiotic-resistant colonies per μg of plasmid

DNA. The object is to determine the mass of plasmid that was spread on the experimental plate and that was, therefore, responsible for the transformants) the number of colonies) observed.

1. 10 μL X 0.005 μg/ μL = 0.05 μg of plasmid

2. 5 μL E. Coli + 250 μL CaCl2 +250 μL Luria Broth+ 10 μL plasmid DNA = 515 μL of total solution

3. (100 μL spread)/(515 μL total solution) = 20/103 fraction of cell suspension

4. 20/103 * 0.05 μg plasmid = 1/103 μg ≈ .00097 μg plasmid in cell suspension spread 5. 1 colony / (0.00097 μg plasmid) = 1.0 * 103 colonies per μg plasmid

What factors might influence transformation efficiency? Explain the effect of each factor that you mention.

Transformation efficiency may be influenced by the amount of plasmid DNA or bacteria that is exposed to one another. Because the amount of plasmid DNA should be proportional to the amount of available bacteria to be transformed, any alteration to this balance can affect the outcome. Procedural errors may affect the result as well- calcium chloride and heat shock effectiveness (or ineffectiveness) can affect the neutralization and entrance of plasmid DNA into the bacterial cell. Heat shock is very specific and calculated, as if the bacteria are exposed to the heat for too long, the cells perish, but if they are left in hot water for too short of time, it is not an effective process. All of these can adversely affect the transformation efficiency in genetic insertion.

Conclusion: Genetic transformation is a very precarious process and must be handled very delicately. The insertion of plasmid DNA into E. coli strains provided some unexpected results that caused us to rethink our original ideas and hypothesize new explanations. Three of the four plates did not glow; the only plate that had luminescent DNA was the plate that was positive for ampicillin. The controls of our group were the agar plates containing ampicillin, because this showed how normal bacteria would grow and reproduce.

Although we were wrong about the ampicillin free agar plate being luminescent, it is possible to see why.

There are some explanations, but the most plausible is the aggregation of cell clusters and the concentration of the bacterial colonies that contained the plasmid for luminescence. However, our predictions of ¾ plates were correct, and luminescence is a very visible trait to record. However, concentrations of luminescent bacteria can affect the overall result-, as too little luminescent bacteria will not be able to be seen.

Transformation efficiency can be affected as well by different factors during the procedure and any limitations placed on the bacteria’s ability to become neutralized or excited in a heated state will adversely affect the results. A new hypothesis can be formulated from these results, but is based on one trial. If the plasmid DNA is transcribed in large amounts, then luminescence will be an obvious trait in a positive ampicillin agar plate. If the plasmid DNA is scarce, then it will not be present in the positive plasmid agar plate.

Works Cited

Watts, S. & Halliwell, L. (Eds.). (1996). Essential Environmental Science: Methods &

Techniques. New York: Routledge.

Redfield, J.K. (1988). Evolution of Bacterial transformation. Retrieved November 21,

Campbell, N. and J. Reese. 2005. Biology. Pearson Education Inc, San Francisco, Ca.

Roe, Bruce A. (December 04, 1997). Bacterial Transformation and Transfection.

Retrieved November 22, 2008 from University of Oklahoma's Center for Genome

Technology: http://www.genome.ou.edu/protocol_book/protocol_adxF.html.