FDH Protein Lab Report

The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

Whenever there is an unknown disease caused by microorganisms, tests are usually made in order to identify the organism causing the disease. There are several tests that need to be made and they include tests such as performing a gram stain, streaking a plate to isolate colonies, inoculating a broth culture, inoculating API strip, and performing oxidase and catalase tests. Having knowledge on how to identify these tests are of high importance in the medical field so it would be to the advantage of those individuals who know how to examine microorganisms and be able to identify it by correctly performing tests on organisms.

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).

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

The selected isolate Gur1 (6 mm disc) was grown on basal salt media supplemented with 2% carboxymethyl cellulose (CMC) at 28º C until substantial growth was recorded (Hankin and Anagnostaksis 1975). The Petri plates were flooded with congo red solution (0.1%), and after 5 min congo red solution was discarded. The plates were then washed with 1M NaCl solution, and allowed to stand for 15-20 min. The clear zone was observed around the colony when the enzyme had utilized by

The pET30a expression vector featuring T7 promoter, N/C-terminal His-tag sequence, T7 terminator and Lac operator was used for ligation of F protein gene at EcoRI restriction sites. The F gene-pET30a ligation was transformed into E.coli strain TOP10 competent cells and spread on Luria Bertani (LB) plates with kanamycin selection. The single colonies were picked and screened for positive cloning through EcoRI restriction digestion. The correct orientation of the gene was confirmed through restriction digestion of the PCR product with ScaI and EcoRV.

Optimum pH determination Enzymatic activity is effected by different environmental conditions; these environmental factors have a direct impact on whether the enzyme will catalyse the reaction tested or if the environment does not permit enzyme activity. One environmental factor that effects enzymatic activity is pH. All enzymes have an optimal environment where the enzyme is most efficient, this environment can even contribute to the function of the enzyme. For LDH the optimal pH would be a pH that results in a rapid conversion of pyruvate and NADH to lactate and NAD+. Based on the experiment the optimal pH of LDH shown in figure 2 is 10.5. This suggests that LDH for conversion of pyruvate to lactate works best in basic/alkaline conditions.

Liquid cultures of the selected bacterial colony were prepared for the PCR amplification of the 16S ribosomal RNA. Two PCR tubes: tube B and tube C, were prepared with each containing 2X PCR Master Mix. Into each tube, 20 µl of 16S RNA primer mix were added. Tube B was selected to contain the prepared bacterial DNA and 5 µl of the liquid DNA was added. Tube C was the selected control tube. It didn’t contain any bacterial DNA and to make up for the absence of the DNA µl, 5µl of sterile water were added to this tube. Both tubes were brought to a total volume of 50 µl. The mixture was kept

In order to achieve this goal they created variants of each gene. These genes were all removed by a similar process by which the pieces of the gene were cut with different restriction enzymes and purified and cloned in order to give us different plasmids. They were amplified using PCR and these were then integrated into the regions and then grown on media and observed. When there was excision of the gene they were looking to remove these strains were cultivated and given designations. Strain WR 441(hDHFR-2) has the hdrA was deleted, WR445 has a deletion of both genes and WR446 (hDHFR-1) has a deletion of hdrB, WR 447 had hts deleted from it. These strains in turn were grown on three different Medias HY which was the rich media, M which was the minimal media and agar plates. In these experiments they found that WR441 needs nothing else and grew on each media with no other enrichment. However WR446 and WR 445 could grow on minimal medium containing both trimethoprim and thymidine, but only when that medium was supplemented with glycine, methionine, pantothenic acid and hypoxanthine. Another strain WR447 was grown and found that it was auxotrophic for thymidine. However when plasmid pHE4 was added to it could grow on minimal medium containing trimethoprim and thymidine. When the hts was removed the hdrB gene did not have a strong promotor which appeared when the hDHFR-2 didn’t express itself enough to give the organism the trimethoprim resistance it need to get a high copy number. Because of this we can use the hdr gene as an expression point for Hf.volcanii. At the lowest level of expression, only complementation of purine, glycine, pantothenate and methionine autotrophy will occur When the gene has a higher level of expression we can see that there will be prototrophic growth in minimal medium, and when the gene is working at its highest level it will give a resistance to

With the new batch of bacteria, a series of biochemical tests were performed. All the biochemical tests performed to identify two unknown bacteria were Phenol Red Broths, nitrate reduction,

By using the ligation mixture formed in experiment 3B, competent cells undergo transformation in this experiment. The cells do not usually exist in a state of transformation ready but they can be made permeable to the plasmid DNA and the capable of transformation are referred as competent. Transformation is the uptake of heritable information in a competent cell. Competent cells care extremely fragile and need to be handle gently to ensure the success of transformation. Water bath is heated to 42oC as heat shock treatment to start transformation. The competent cells containing recombinant vectors can be identified through blue white screening. Cells containing recombinant vectors are able to survive on Luria Bertani (LB) medium supplemented with the antibiotic, penicillin. The cells transformed by recombinant vectors are plating on plate that containing isopropyl β-D-thiogalactopyranoside (IPTG) which as an inducer of the lac promoter, and 5-bromo-4-chloro-3-indolyl- β-D-galactoside or called X-Gal which is a dye that produces a blue colour when

RHA1 genome contains numerous genes involved in biphenyl degradation. The biphenyl pathway was confirmed by the discovery of genes that encode necessary enzymes for biphenyl metabolism. As reported by (Furukawa and Miyazaki, 1986; Taira et al., 1992), bph operon was first cloned from the bacterium Pseudomonas pseudoalcaligenes KF707. Since then, bph genes from several Gram-positive and Gram-negative bacteria have been cloned, and shown to be present on bacterial chromosomes, plasmids and transposable elements (Nishi et al., 2000).

We selected the universal primers based on the highly conserved NADH dehyedrogenase 1 gene (Bowles and McManus, 1993) and our data indicated that the purified and partially sequenced PCR products generated 399 bp of NADH dehydreogenase 1 gene. The sequances were aligned by cluster grouping where the clusters aligned the most similar sequances firstly then progressively more distant groups of