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11	Biochemical Study of Engineered Fluorescent Proteins as Calcium Sensors and the Effect of Calcium and PH in Cell Reproduction and Protein Expression Delgado, Malcom Arturo 01 December 2009 (has links) Calcium plays important roles in both eukaryotic and prokaryotic cells. Its actions help to stabilize cell synthesis, growth and development. In this thesis, studies have been completed to determine effects of calcium and pH on bacterial cell growth and protein expression using the bacterial cell strain E.coli BL21(DE3). Our studies demonstrated the addition of calcium addition in the media does not affect growth but increases protein expression, while reducing the pH from 7 to 4 through the addition of 10mM EGTA in LB media inhibits both. Additionally, we report studies on the design, expression, and purification of fluorescent mCherry variants and their differences in their optical properties, including: extinction coefficients , quantum yields and pKa values. Also, we report progress in the crystallization of two GFP calcium sensors: G1 and D1, using 13 and15% PEG 4000 and 3350 respectively in 50mM HEPES buffer (pH 6.8-7.0) in an effort to optimize crystallization. Calcium Protein mCherry Green Fluorescent Protein (GFP) E. coli. X-ray crystallography Protein expression Fluorescent proteins BL21(DE3).
12	Structural Flexibility and Oxygen Diffusion Pathways in Monomeric Fluorescent Proteins Regmi, Chola K 26 March 2014 (has links) Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins. Fluorescent Protein Chromophore mCherry Oxygen diffusion Protein barrel Structural flexibility Photostability Free energy Molecular dynamics Biological and Chemical Physics
13	The use of Gibson Assembly for DNA cloning / Användning av Gibson Assembly för att klona DNA Johansson, Samuel January 2022 (has links) This thesis report revolved around the cloning process of plasmids. Attempts of cloning the red fluorescent protein mCherry, and the green fluorescent protein EGFP from various plasmids, into other plasmids containing different cell-junction/cytoskeleton plasmids were made. These plasmids were first amplified using PCR, and then cloned using Gibson-Assembly, and then transfected into live HEK293T or MDCK-II cells. After the transfection, the cells were examined in a microscope. The results showed no signal or localization for the cloned plasmids in their respective corresponding channel, 561 nm for the red fluorescent protein mCherry or 488 nm for the green fluorescent protein EGFP. The step that went wrong was the PCR step in the cloning process, since the backbone vector was not successfully amplified. The reasons for this was either that the backbone vector was too long, the primers regions were to rich with Guanine and Cytoseine, or the primers being too long. / Den här tesen kretsade kring kloningsprocessen för plasmider. Det gjordes försök att från plasmider klona in det röda fluorescerande proteinet mCherry, samt det gröna fluorescerande proteinet EGFP in i andra plasmider som innehöll olika cell-junction proteiner. Både det fluorescerande fragmenten och plasmid-vektorerna innehållande cell-junction proteinerna amplifierades med PCR. Sedan gjordes Gibson-Assembly som var själva kloningsmetoden. Efter det transfekterades HEK293T, samt MDCK-II celler med lösningen från Gibson-Assembly kloningen. Dessa celler undersöktes sedan i mikroskop. Resultatet visade inga tydliga signaler varken i 561 nm kanalen (mCherry), eller i 488 nm kanalen (EGFP), vilket betyder att kloningen inte fungerade. Steget som gick fel var PCR-steget i själva kloningsprocessen, då plasmid-vektorerna inte amplifierades. Anledningen till detta var antingen att själva plasmid-vektorerna var för långa, primer regionerna hade för mycket Guanin och Cytosin, eller att alla primers själva var för långa. Plasmid Primer Cloning PCR Gibson-Assembly Transfection Fluorescent protein Fluorescent imaging mCherry EGFP Zona-Occludens 1 Occludin alphaTubulin Cx-43-7 Plasmid Primer Kloning PCR Gibson-Assembly Transfektion Fluorescerande protein Fluorescerande avbildning mCherry EGFP Zona-Occludens 1 Occludin alphaTubulin Cx-43-7 Physical Sciences Fysik

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