Results The experiment conducted was to determine the expression of the lac z gene under different treatment conditions. The first treatment was to add 0.3% glucose to the E. coli. The second was to add 0.2% lactose. The third treatment was similar to the second one, in which 0.2% lactose was added, except at a later time. The only difference between these trials is the time the lactose was added. Treatment four was adding 0.3% glucose and 0.2% lactose to the E. coli. After the data was gathered, the treatments were analyzed through absorbance and the time required for reactions to take place. Table 1 summarizes the information about the bacterial growth, such as the OD600 reading shows how bacteria grew in each sample during the incubation period. As the figure indicates, the samples that contained glucose had a higher OD600 reading, which indicates that the bacteria samples grew much better in glucose rich environments compared to the environments containing lactose. Also, the samples that have higher Abs420 readings for less reaction time indicate that there is a higher presence of active β-galactosidase which can interact with the ONGP. The information in Figure 1, which was obtained through a β-galactosidase activity assay, indicates that the samples with lactose and no glucose have the higher activity level for β-galactosidase, where the samples that did not have the lactose added later were able to have a higher reaction rate with the ONGP. The amounts of β-galactosidase present in the four samples is confirmed by the results of a Western blot, shown in Figure 2, which indicates that there is a higher level of the protein in those samples, and it also indicates that there is little to no β-galactosidase produced within th... ... middle of paper ... ...ny qualities that make it an easy protein to detect (2009). This could also be used in many other transgenic experiments by using it as a selectable marker, where only the cells that have it could survive in lactose, or by viewing it as a gene that loses its ability to function after the gene insertion where the lack of β-galactosidase would indicate that the gene of interest was correctly inserted. Works Cited Forss-Petter S, Danielson PE, Catsicas S, Battenberg E, Price J, Nerenberg M, Sutcliffe JG. 1990. Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control. Neuron 5(2): 187-97 Debacq-Chainiaux F, Erusalimsky J, Campisi J, Toussaint O. 2009. Protocols to detect senescene-associated beta-galactosidase (SA-β gal) activity, a biomarker of senescent cells in culture and in vivo. Nature Protocols 4: 1798-1806
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
For example, incubating the samples at different temperatures would create more data points to establish an optimal temperature. From the results in the experiment in this study, it is known as temperature increases, enzymatic activity increase, and vise versa. However, what can not be observed is at what point does the increase in temperature begin to denature the enzyme, above 60°C. Furthermore, assays can be preformed to determine optimal pH, as well. From Dutta’s, and his partners, experiment it shows that there is a range where the Heliodiaptomus viduus’s lactase shows the most activity, which is between 5.0 and 6.0
For example, if a person had been able to consume lactose products for their life with no problems, but in an unfortunate event had to have a portion of his or her small intestine removed, there would be a change in the number of present lactase enzymes in the stomach. Because the lactase enzyme is stored in the small intestine, the person may now experience lactose intolerance due to the decrease in the presence of lactase. Knowing where the lactase enzyme is stored can aid physicians in understanding what will happen after a procedure or the introduction of a new medication. The experiment was conducted to determine the optimal ph of lactose required to produce the maximum amount of glucose. It was predicted that the optimal ph of lactose would be most efficient at lactose ph 6, and that the lower the ph, the amount of glucose produced would increase
The green fluorescent protein (GFP) gene is a naturally occurring gene from a bioluminescent jellyfish. The gene allows for objects and animals to glow in the dark when activated by the presence of the sugar arabinose in the pGLO plasmid. The GFP gene is often used as a marker for gene expression and genetic transformation. The pGLO plasmid is a genetically engineered plasmid used as a vector in biotechnology to generate genetically modified organisms(GMO). M. Chalfie et. al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things.
We can measure the amount of beta galactosidase produced in each tubes indirectly although it is difficult. ONPG is converted to galactose and o-nitrophenol by beta-galactosidase which has a yellow color with an absorbance at 414nm. The amount of ONPG
Enzymes are proteins that increase the rate of chemical reaction by lowering their activation energy. The enzyme glucose oxidase is one of the most widely used enzyme as an analytical reagent due to its ability to identify the presence of glucose, its low cost and good stability. This report discusses the role of enzymes concentration in biological reactions and the catalytic activity of glucose oxidase on D-Glucose. The activity was studied by spectrophotometry and the results were first tabulated and then plotted. The results of this experiment indicate that the enzyme concentration has no major affect on the rate of
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
E. Coli will be used as an example of how an inducible operon works. E. Coli's main source of nutrition is glucose. If glucose is not available, it can utilize lactose. But the necessary enzymes used to digest lactose aren't normally made by E. Coli. When lactose is available in the environment, certain activities will take place allowing E. Coli to digest lactose. Lactose induces transcription of enzymes to utilize lactose.
While the Type I Gaucher Disease is non-neuronopathic (not affecting the nervous system) the second two types are neuronopathic. Yet even though the three types of Gaucher produce different symptoms, all three types result from the same cause: a lack of glucocerebrosidase enzyme. The glucocerebrosidase enzyme functions to break down the compound glucocerebroside, a fatty compound which usually is stored in all cells of the body in very small amounts. In Gaucher patients, an excess of glucocerebroside builds up in the body, and is stored abnormally in lysosome, or storage cells (3) . Typically, macrophages are able to aid in the degradation process of glucocerebroside. However, due to the lack of glucocerebrosidase in Gaucher patients, glucocerebroside stays in the lysosome, preventing macrophages from acting upon them. Macrophages which are enlarged and contain an abnormal buildup of...
In this experiment, in the first part, the best concentration of enzyme was determined by recording the absorption over time. In the second part, the best concentration was selected from the previous experiment which was C and the optimum pH was determined.
Carbohydrate Utilization: Two culture tubes, phenol red lactose broth and phenol red sucrose broth, were each inoculated with one loopful of organism 3 from a broth culture. The broths were incubated at 37°C. After 24-48 hours, the mediums were examined. A positive test result is indicated by a change from the red broth to a yellow broth. A change from red broth to orange broth, or no change in color, is indicated as a negative result. A gas bubble produced in the Durham tube is indicated as a positive result for gas. A negative result for gas is indicated by no gas in the Durham tube.
This lab attempted to find the rate at which Carbon dioxide is produced when five different test solutions: glycine, sucrose, galactose, water, and glucose were separately mixed with a yeast solution to produce fermentation, a process cells undergo. Fermentation is a major way by which a living cell can obtain energy. By measuring the carbon dioxide released by the test solutions, it could be determined which food source allows a living cell to obtain energy. The focus of the research was to determine which test solution would release the Carbon Dioxide by-product the quickest, by the addition of the yeast solution. The best results came from galactose, which produced .170 ml/minute of carbon dioxide. Followed by glucose, this produced .014 ml/minute; finally, sucrose which produced .012ml/minute of Carbon Dioxide. The test solutions water and glycine did not release Carbon Dioxide because they were not a food source for yeast. The results suggest that sugars are very good energy sources for a cell where amino acid, Glycine, is not.
The mixture for that table’s flask was 15 mL Sucrose, 10 mL of RO water and 10 mL of Yeast, which the flask was then placed in an incubator at 37 degrees Celsius. In my hypothesis for comparison #4 the measurements would go up again with every 15 min. intervals because of the high tempeture and also be higher that then Controlled Table’s measurements. Hypothesis was right for the first part but was wrong for the second part of the comparison, the measurements did increase in the table’s personal flask but the measurements did not get higher than the Controlled Table’s measurements, see chart below. In conclusion, I feel that the substitution of glucose for sucrose made the enzymes work just as hard as the Controlled Table’s flask but just not as much because sucrose was too strong for the enzymes to
Gene therapy is a provisional technique that is the insertion of normal genes into the cells where there is a missing or miscoded gene to fix a genetic disorder. In the 1960s and early 1970s,
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.