Method:
- Add 5cm³ sodium phosphate to four test tubes.
- Transfer 1cm³ E. coli in nutrient broth to the first test tube, and 1cm³ E. coli plus nutrient broth (lactose) to the third.
- Add ß-galactosidase to test tube four.
- Add 5 drops methylbenzene to each test tube, and 1cm³ ONPG to each test tube. Bung all tubes and shake to disperse all chemicals.
- Place all test tubes in a 35°C water bath and leave for at least 1 hour.
- Place a sample from each test tube into a colourimeter and record results.
Reasons for particular equipment/method:
- To prevent cross-contamination and the resulting ruination of experiment.
- Safety of all people involved, to avoid poisoning and irritation.
- Methylbenzene (toluene) is used to break down the membrane of the cells of the bacteria, allowing the lactose access to the genetic material, which is free inside the cell.
- The sodium phosphate acts as a buffer to ensure the best pH for the enzyme to work in once it has been induced.
Results: Table to show optical density of four samples of prepared mixture after 1 hour in a water bath:
Graph on separate sheet
Analysis:
The enzyme needed by many organisms to be able to digest lactose is beta-galactosidase. This enzyme catalyses the reaction that results in lactose being broken down into galactose and glucose. Lactose is a disaccharide, a type of sugar that is actually made up of two sugars – monosaccharides that are only made up of themselves. The two sugars that make up lactose are joined by a condensation reaction resulting in a glycosidic link. Here is a diagram of the breakdown of lactose; you can see the two monosaccharides glucose and galactose, and the glycosidic link:
This reaction will not happen alone, it needs the enzyme beta-galactosidase to induce it.
But, the bacteria E-coli does not have this enzyme operating normally in it’s metabolic system, although it does have the genes to code for it; these are locked. The particular gene needed is prevented from being transcribed by RNA polymerase by a repressor molecule, which is coded for by a regulator gene that is always switched on. The repressor molecule (a type of protein) is usually bonded to the section of DNA which will result in the production of beta-galactose if coded for. If lactose is present, however, instead of somehow switching off the gene that produces the repressor molecule, as would be expected, it binds to the repressor molecule itself, drawing it away from the DNA and leaving it free to be transcribed.
Because only one molecule of lactose is needed per cell to switch on the gene for beta-galactose, there is still plenty left to be digested and the resulting two monosaccharides used in respiration for energy.
In this way, the lactose is acting as a biological catalyst:
In our experiement, we added lactose to the food source or a bacteria, fully aware that it was not at that time capable of digesting it. Once we added lactose, the above process of unlocking the gene for beta-galactose was activated and beta-galactosidase produced, which meant that the lactose could be digested.
The chemical ONPG (Orthonitrophenyl-ß-O-Galactoside) is another chemical that can be catalysed by ß-galactosidase. It is a colourless compound, but when borken down by the enzyme, the products are galactose and ONP, which is a yellow liquid. Therefore when we add this to our mixtures in which we hope to produce beta-galactosidase, ONP will also be produced because the ONPG is in contact with the enzyme. This will be a visual indicator that ß-galactosidase has been induced.
Evaluation:
Our experiment was successful in that we managed to produce beta-galactosidase, evident by the yellow colour of test tube three which contained lactose and ONPG. Unfortunately, we must have contaminated test tube two with beta-galactosidase, as this should not have turned yellow as we were not meant to add beta-galactosidase to breakdown ONPG into yellow ONP. The reason test tubes two and three had such similar APS values is because they were essentially identical, containing ONPG and lactose, and reaulting in the formation of ONP. Therefore if I were to repeat the experiment I would ensure that we were very careful to prevent cross contamination by being much more meticulous in our chemical transfers and aseptic techniques.
Test tube one was also somehow contaminated, this time with lactose – I can tell this because although it contained ONPG and e.coli, it should not have contained the lactose which began the pathway of chemical reactions that resulted in the breakdown of ONPG and the yellow colour.
Therefore, although there is nothing wrong with method used, we were not careful enough in prevention of cross contamination and the results were skewed because of it. If we had been much more particular in our manner of carrying out the experiment, it would have given much more understandable and clear results.
Conclusion:
In conclusion, our results did match the hypothesis in that we managed to induce the production of beta-galasctosidase, and saw the resulting breakdown of ONPG and therefore also the breakdown of lactose into galactose and glucose, although we can only presume that this part of the experiment was successful as we had no way of measuring it. Unfortunately, some of our results were not as predicted because of the inprecise way we carried out our method, and two of the controls were distorted.
Bibliography:
http://vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/lacOperon/
http://www.blc.arizona.edu/courses/181gh/rick/expression1/inducible.html http://138.192.68.68/bio/Courses/genetics/lac%20lab/laclab.htm
http://www.biology.ucsc.edu/classes/bio105l/EXERCISES/ASSAY/handout.pdf
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