β galactosidase activity Lab Report by Ruchit Shah PDF

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β-galactosidase assay using Statistical Analysis Submitted by – Ruchit Shah

Part A: Data presentation and description 1. Mean β-galactosidase activity (in Miller units) with standard deviation was obtained using

JMP application. Fig. 1 Comparison of mean β-galactosidase activity of untransformed E.coli DNA, pREP4A DNA & pREP4B DNA plotted against Water, Lactose & Lactose + Glucose samples. The purpose of this

graph is to determine the levels of beta galactosidase activity in the untransformed, pREP4A and pREP4B DNA. The above mean values were obtained through statistical analysis in JMP 15 application. The connecting letter groups A reports that the untransformed E. coli DNA and pREP4B DNA had the highest levels of β-galactosidase activity since the concentration of lactose is higher. Group B reports that the untransformed E.coli DNA, pREP4A DNA and pREP4B DNA in the presence of Water and Lactose + Glucose showed lowest levels of β-galactosidase activity. In the case of lactose present in pREP4A DNA, the β-galactosidase activity is low since pREP4A DNA is the functional copy of plasmid DNA that inhibits the expression of genes involved in metabolism of Lactose.

Part B: Interpretation 1. The results of the β-galactosidase assay presented in the above figure indicates that the pREP4A DNA with Lactose + Glucose sample had the lowest levels of β-galactosidase enzyme. Yes, it is expected as in this low level of transcription occurs. If both glucose and lactose is present, β-galactosidase synthesis is not induced until all the glucose has been used up. After all the glucose is utilized, the lac operon operates to utilize the lactose present. Since an inducer (allolactose) is present, the lac repressor is released from the operator. But due to low levels of glucose present the cAMP levels are also low. Thus, the Catabolite Activator Protein (CAP) is inactive and cannot bind to the DNA, the transcription tends to occur at lower levels.

2. Based on the DNA-sequencing and agarose gel electrophoresis in the past experiments showed that pREP4A plasmid would be best choice to use in further research because it being the functional plasmid, the lac repressor will prevent the transcription of gene involved in lactose utilization. This will help researchers to insert special genes and the plasmid would be more accurate while it only needs glucose to metabolise.

3. (i) β-glucan assay– β -glucanase is an enzyme that hydrolyses beta-glucans. It is widely used in brewing products like beers. The most important enzyme found in barley is endo-β→3, 1→4-glucanase which initiates the hydrolysis of β1→4 linkage adjacent to a β1→3 linkage, hence resulting in rapid reduction in the viscosity of β-glucans. This enzyme is particularly absent in raw barley, but is synthesized in abundance during germination. This enzyme is broken down with the help of beta-glucanase and hence it increases the function of malt production in the barley. (Hrmova and Fincher, 2001) (ii) ⍺-glucosidase assay – ⍺-glucosidase assay is a colorimetric assay for determination α-glucosidase in human semen samples. Starch is converted to simple sugars when glucosidase enzyme catalyses the starch present. ⍺-glucosidase works by competitive and reversible inhibition of intestinal enzymes. (Tomasik and Horton, 2012).

4. The advantages of having such system where single cell is transformed with pREP4 plasmid is that they have system that ensures the recipient cell does not contain the similar element. The genetic transfer is beneficial to recipient plasmid that may include xenobiotic resistance, antibiotic resistance or the ability to use new metabolites. It is difficult for E.coli to breakdown lactose. LacI gene inhibits the mRNA production of proteins encoded by lac operon. Since, the lac operator (LacO) overlaps the promoter, the LacI gene stops the RNA polymerase to further bind to the region and encode lacZYA genes. The LacI . LacO association occurs when there is no lactose present. Hence when lactose undergoes confirmational changes it is converted into allolactose, this helps to release the operator and thus RNA polymerase is freed that eventually generates numerous copies of mRNA encoding the lac enzymes.

References Hrmova, M., and Fincher, G. B.Structure-function relationships of β-D-glucan endo- and exohydrolases from higher plants. Plant Molecular Biology 47 (2001): 73–91.

Piotr Tomasik, Derek Horton, in Advances in Carbohydrate Chemistry and Biochemistry, 2012....