Essays on Lab Report Assignment

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Table of ContentsIntroduction. .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .Green Fluorescent Protein (GFP)Expression of proteins as Glutathione-S-transferase (GST) fusion proteinsStatement of the PurposeAim of this ExperimentMethods. .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ... Day 1Setting up the PCR screen and streaking sampled colonies Agarose gel electrophoresis of PCR productsRestriction endonuclease digests of pGEX-GFPS65T-1, 2 and 8Fluorescence scoring: GFPS65T-1, 2 and 8Day 2Agarose gel electrophoresis of restriction enzyme-digested plasmidscDNA sequence analysis: pGEX-GFPS65T-1, 2 and 8Purification of GST-GFPFluorescence scoring: streaking E. ColiCollation and interpretation of individual and class dataThrombolytic cleavage of GST-GFPCollated class: PCR and fluorescence analysis E. coliThe size calculation of restriction digest products using the plasmidDay 3Thrombolytic cleavage of GST-GFP (cont)SDS-PAGE analysis of bacterial lysate, GST-GFP and GFPSpectrophotometric measurement of GFP concentration (A280) using EBradford protein assayCoomassie blue protein stainingCalculation of the molar extinction coefficient for GFP using “protein calculator”Spectrophotometric measurement of GFP concentration (A280) using EBradford protein assayResults. .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .Day 1Day 2PCR and fluorescence analysisGel image of the PCRTotal yield of GFP from the purification processDay 4RR for the non-induced sampleRR for the induced sampleDiscussion. .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ... Day 1 & 2Day 3Day 4CDNB assay for GST activityRR for the induced sampleSummary of the Lab Work. .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ..Introduction In this practical the focus is upon one protein, that of GFPS65T.

In this practical we are going to typify the expression vector pGEX-GFPS65T. This vector sets a fusion protein of GST and GFP-GFPS65T. Purification of this protein will then be done by affinity chromatography after which it will be cleaved using thrombin for the production of a pure preparation of GFP. Green Fluorescent Protein (GFP)The green fluorescent protein (GFP) is a protein made up of 238 amino acids. When blue light is shown on it, it displays bright green fluorescence (Prendergast & Mann, 1978). GFP gene is made use of in cell and molecular biology.

In this field the use of this gene is as a reporter of expression (Philips, 2001). With a little modification, GFP has been used for making biosensors. Several animals have also been formed through which there is the proof-of-concept of GFP that there can be the expression of a gene in the whole organism. The introduction of GFP gene can be done in organisms and their maintenance follows in their genome. This maintenance is by means of breeding, injection using a viral vector, or through cell transformation.

There has been the introduction and expression of the GFP gene in several bacteria, yeast and different fungi, fish, plant, and mammalian cells, which also includes human. In GFP the amino acids 65-67 have the florescent chromophore (Cheng, et al. , 1996). These amino acids are serine-tyrosine-glycine in the case of the wild-type protein. A bright green light is emitted from the wild-type GFP which has λmax = 508nm. This emission takes place when the GFP has been excited with UV light (λmax = 395nm).

However, in this case the excitation goes away soon with time (Cheng, et al. , 1996). When the wild-type protein is excited with blue light (λmax = 470nm), it fluoresces stably, however, the fluorescence is weak. Because of such reasons there was a difficulty is detecting the wild-type GFP protein in plant and mammalian cells, and so there was not much application of them in these mechanisms.

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