Hypothesis:
By adding lactose to the culture of E. coli, this will introduce the need for Beta-galactosidase in the bacteria. The presence of lactose will overcome the forces holding the repressor molecule to the gene which will code for the amino acids which will form the enzyme, therefore the RNA polymerase will be able to bind to the gene and cause the DNA to unwind, allowing mRNA to be produced that codes for the genes. Once the enzyme beta-galactosidase is produced, it will break down the disaccharide lactose into the monosaccharides glucose and galactose. Therefore in the culture of E. coli in nutrient broth with lactose the ONPG added will produce ONP turning the solution yellow, indicating that beta-galactosidase has been formed. Conversely, I expect the culture of E.coli in nutrient broth without lactose to remain turn only slightly yellow as there is no lactose to switch on the gene and produce the enzyme.
Plan:
In order to test out my hypothesis, I will use four different solutions. The first will contain a culture of E.coli in nutrient broth without lactose, and the second will contain a culture of E.coli in nutrient broth with lactose. I will be able to compare these two test tubes directly to see the effect of lactose on the E.coli. The third test tube will contain 1cm of distilled water, I will use this test tube as a control, and this will help ensure that my results are reliable. In the fourth test tube I will add beta-galactosidase; I expect this solution to turn yellow immediately as the enzyme is already present. By using four different solutions this will allow me to compare my results and the control will allow me to identify any errors. I will carry out the experiment using aseptic techniques as outlined in my safety procedures as any contamination of the bacteria may affect my results. I will leave the test tubes in the water baths for approximately 10 minutes and then record my results in a suitable table.
Apparatus:
Safety Precautions:
Aseptic techniques should be employed when using microorganisms so there in no risk of the student becoming infected or cross contamination. Firstly we wore gloves and washed our hands thoroughly. Before we set up the apparatus we made sure the working area was clean by wiping it with disinfectant. The disinfectant used may be toxic or harmful and likely to irritate the skin, so again gloves must be worn. We set up a bunsen burner around our working area so any bacteria in the air will rise and not fall into any of the test tubes. When transferring the E.coli we made sure to use sterile pipettes to avoid cross-contamination. In addition when opening the flask containing E. coli in nutrient broth we briefly held the top of it in the flame, to kill unwanted microbes and then sterilized the top of the tube or flask again before immediately closing it. It is also important to hold bottles and tubes at an angle to minimize the amount of airborne microbes that can fall into them.
In addition methylbenzene is highly flammable and harmful by inhalation, so when using you should keep it away from flames. The vapour is irritating to eyes and mucous membranes so it should be used in a fume cupboard whilst wearing goggles.
Ethical Issues:
Using bacteria in an experiment may highlight many ethical issues. Bacteria are considered living organisms; therefore some may consider it wrong to endanger their lives by using them for scientific research. However it is far more ethical to use bacteria than using humans or other animals. Therefore I feel that the results of the experiment outweigh the ethical issues because of their ability to quickly grow and the relative ease with which they can be manipulated, bacteria are the most suitable living organism to use.
Results:
Analysis:
I predicted that in the presence lactose, beta-galactosidase is produced, enabling the cells to use lactose as a source of energy for growth. My results prove this hypothesis to be correct. Test tube 2 turned yellow within a few minutes indicating that the lactose overcame the forces holding the repressor molecule to the gene which coded for the amino acids which formed the enzyme beta-galactosidase. Once the beta-galactosidase was produced, it broke down the disaccharide lactose into the monosaccharides glucose and galactose. Therefore the presence of beta-galactosidase turned the ONPG compound into a yellow compound of ONP. I also predicted that the culture of E.coli in nutrient broth without lactose will only turn slightly yellow. My results again prove my hypothesis correct as the solution in test tube 2 was a pale yellow, this was due to the absence of lactose so the gene was not able to be switched on. In addition the test tube containing beta-galactosidase at the start turned a dark yellow as the enzyme was present at the start so the production of yellow ONP happens much quicker and therefore creates a darker colour. Test tube 3 remained colourless which showed that the control worked efficiently.
Evaluation:
I think that overall my results were quite accurate, although there were a few areas which could have affected the results. It was very important to carry out aseptic techniques. I ensured that when transferring the E.coli I used sterile pipettes to avoid cross-contamination. When opening the tube or flask containing the E.coli nutrient broth I made sure I briefly held the top of it in the flame, to kill unwanted microbes then sterilized the top of the tube or flask again before immediately closing it. In addition it is important to hold bottles and tubes at an angle to minimize the amount of airborne microbes that can fall into them. However there was still a slight risk of cross contamination. Despite the use of a Bunsen burner bacteria could have still fallen into the test tubes which would have affected my results and made them unreliable. In addition bacteria from overalls and any present on the gloves may have also contaminated the solutions. In addition the amount of time the test tubes were left in the water bath could have affected my results, as if they were not in there for long enough we would not have been able to see the final result. If I was to carry this experiment out again I would measure the colours of the solutions more accurately by using a spectrophotometer or colorimeter to measure the amount of ONP formed in a given time. This would of given more accurate results and I would have been able to plot the numerical data on a graph which would allow easier comparison. Overall I thought my experiment worked quite well as I successfully proved my hypothesis.
