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EDVO-Kit it 222 Transformatiori of E. coli with pFIuoroGreen™ and pFluoroBlue™

Bacterial Transformation

Bioluminescence of marinę microorganisms has been observed by many summer visiłcrs at becches around the world. Onlookers are always fascinated by fhe rcpeated paradę of color and light on fhe sand during the ebb and flow of Ihe tice. This observa:k>n pcles to fhe light produced by fhe bioluminescent jelly fish Aquorea victoria. the natura! host of the green fluorescent protein (gfp). A bright bursl of light^- is observed when energy ;s transferred to the green fluorescent orctein {gfp) located in specializcd photogenic cells in the base of fhe jelłyfish urr.brella. 5everal variants of the gfp protein have been engineered and used to dramcti-cally enhance classrcom labcratory experiments. An excellent compan-ion to gfp is the genelically engineered ande well-cnaracterized blue fluorescent protein (bfp).

fhis fam.ily of proteins has been known for some tirne and significant rcsearch in this area has been reported. Fluorescent proteins can be expressed both in prokaryotic and eucaryotic cells. These proteins do not require substrates. other gene products, or cofactors. When exposed to long U.V. light, they emir a bright green or blue fight that is visibie in bacleria Iransformed by piasmids that contain the genes ecoding gfp or bfp. Likowisc, purification of the gfp or bfp from crude protein extracts is simpiified by their fluorescent detecticn.

In celi biology experiments. the bfp ane gfp proteins are often fused to other proteins to study various biochemical processes. There are many examples of these chimeric proteins. fusion products using the gfp or bfp fluorescent proteins as biological tags. Such fusions are at either the N or C- terminal cnd usualiy ■•esult in no biological cctivity toss in the protein under study or in bfp or gfp fluorescence. These chimeric proteins can be used as biotechnological tools, allowing studies of pher.omena such as celt cycle and protein localization and trafficking within cells.

The green fluorescent protein (gfp) posscscs 238 amino acid residues and has a molecular weight of approximately 40,000 daltons. Most of the intact protein is required for maintaining fluorescence; only smali deletions of a few amino acids are allowed without compromising the integrity of Ihe protein structure. Interesłingly, the chromophore responsible for light emission is wiłhin the primary struć furę of the protein and resides in posi-tions 65 to 67, a cyclic tripeptide composed of the amino acids Ser-Tyr-Gly. The importance of protein folding is clearly demonsłrałed in that the gfp is fluorescent only upon proper and fuli confcrmafional folding.

The blue fluorescent protein (bfp) is a derivałive variant of fhe gfp with a His ło Tyr substitution at position 66. A second bfp derivative possessses a second substitution from Tyr to Phe at position 145. The initial brp, known as P-4, had only the His-óó substitution and is not as bright as the double mutant. With fhe crystal slructure of gfp being determined. several other variations of Ihe gfp could well be constructed using site-directed mu-łagenesis (SDM). SDM allows specific (point) mutations to be iniroduced

Background Information

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