Glutathione's Transferase Lab Report

Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.

This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal

RNA polymerase can bind to the promoter region of the DNA even when the lac repressor is bound to the operator site.

The addition of 5-bromo-4-chloro-3-indoyl-β-thiogalactoside (X-gal) in the agar mix acts as an artifical substrate for the enzyme so it produces blue E.coli colonies when hydrolised by the β-galactosidase meaning those specific colonies do not contain the plasmid with the CIH-1 insert (non recombinant E.coli) (Coventry University 2016). Isopropyl-β-D-thiogalactoside (IPTG) an artifical transcription inducer of the lacZ gene is also added into the agar mix, binding to the repressor gene and inactivating it. Therefore when the CIH-1 gene is incorporated within the plasmid MCS and inserted into the E.coli

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

Using E.coli for the induction of Beta-galactosidase within the lac operon upon a variety of conditions. Jenna Riordan (9, Oct. 2017) Introduction The lac operon has answered many questions about gene expression over the past two decades. This mechanism has also helped scientist understand interactions between proteins, proteins-genes, and protein-DNA.(Hediher, et al., 1985)

In analyzing the effect of 2-aminopurine on mutation frequencies of bacterial strain CC102 (ara∆(gpt-lac)5 thi rpsL/F’ lacZ-Y+A+proA+B+ , the bacterial strain CC102 was cultured in LB medium with 2-aminopurine and one LB medium without 2-aminopurine. The bacterial strain CC102 with and without the 2-aminopurine mutagens were diluted to 10-5 and 10-6 and were plated on LB plates with nutrients and sources of carbon compounds for growth. The undiluted bacterial strain CC102 was then plated on minimal medium with thiamine and lactose. In noting the genotype of the bacterial strain, there is a deletion in the lac gene as it is ∆(gpt-lac) and the lacZ gene is not functional, therefore β-galactosidase is not able to cleave lactose into the constituents of glucose and galactose. In table 1.1, when observing the number of mutant colonies on minimal lactose and single cell colonies formed on LB plates with no introduction of the 2-AP mutagen, the mutant colonies were not formed on the minimal lactose yet were formed on the LB nutrient rich plate. For example in looking at group 6 data, 163 colonies were formed on LB medium at 10-6 yet no colonies were formed on minimal lactose with no 2-AP. Since the lac gene and the lacZ gene need to be functional to cleave lactose, it would allow for growth on the minimal medium with lactose but since there was a deletion for the lac gene and lacZ gene was not functional, no growth occurred. However with the introduction of the

The testing of various proteins was performed by comparing the molecular weight of proteins using SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy of structure that consists of folding that determines the direct function of each protein.. The molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the protein structures and causes them to separate due to the change in bonding charge. SDS and heat are how the proteins are denatured. The process of denaturing a protein is breaking

Through multiple experiments, promoter elements were examined to see how they control gene expression. The purpose was to learn how a gene is regulated in bacteria, how timing affects activity, how positive and negative regulation control gene expression, how E. coli mutants respond in plate assays, and how concentrations of sucrose affects gene expression. ß-galactosidase enzyme, also known as Beta-gal or ß-gal, was the assay in the experiments. ß-galactosidase is an enzyme that breaks up lactose into galactose and glucose. Furthermore, it is a hydrolase enzyme that catalyzes the hydrolysis of ß-galactosides into monosaccharides.

In order to manipulate, clone and express certain genes it is essential that a greater understanding of how the gene in question operates, in order to isolate and potentially recreate that particular characteristic in a host organism (Strand ect al. 2003) Escherichia coli is almost the most used organisms of preference in lab based experumentation for the manufacturing of recombinant proteins. Its used as a mobile factory because it is stable and it has become the most famous expression platform organism to use. (Lee ect al.

Spectrophotometer; the finding of protein concentration of an unknown sample of BSA, and by using the standard curve.

The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally. Throughout this experiment, CTAB was used to kill the E. coli celles at specific time sets so that the cell lyses and releases its content. This is very important as if the cells do not lyse, the beta galactosidase would remain in the cells and there would be no way to measure the amount of beta galactosidase produced. We can measure the amount of beta galactosidase produced in each tubes by knowing the

Light was also created when revealed to horseradish peroxidase and this lead to the light being shown on photographic film. Other detection methods include protein staining with Coomassie Blue which for example, provides an image of the proteins shifted onto the membrane. In addition, it was noted that failure may result if denatured proteins are unable to form interactions and can even result with exchanges with nonspecific proteins by proteins in non-native conformations. Also that denaturing likely affects prey proteins more. Moreover possible problems that could arise in this method is that there could be incomplete transfer or even too many bands seen on the blot. The solution for the latter would be to check the antibody with the western blot standard or to reduce their concentration/incubation length. Taken as a whole the far western blot prospers when it resembles the western blot