HaroonSaleem.docVIP

Haroon Saleem Bio 212AB GFP Isolation Introduction: Green florescent protein was first isolated from the jellyfish Aequorea victoria. With the cloning of the gene for GFP from A. victoria the possibility has risen of producing the protein heterologously in large quantities. GFP has the advantage that is glows a brilliant green under long wave UV light and can be used to study cells at the molecular level or track a particular protein and can be as a biomarker. GFP has been shown to form without the need for any cofactors or specific posttranslational processing. Aequorea GFP exhibits two absorbance peaks at ? = 395 and ? = 475 and has an emission peak at 509 nm. Materials and Methods: Cell culture: E. coli cells that had been transformed using the pGLO plasmid by the Bio 212AA class had been obtained and 6 distinct colonies was picked and transferred to 6 15 ml centrifuge tube containing 5ml LB broth Ampicillin and L-arabinose. The amounts of Amp and L-arabinose added will be discussed later as this small broth was pipetted from a 500ml culture broth. The centrifuge tubes were incubated in the floor shaker for 16 hours at 32°C. The optimum folding temperature for GFP is 32°C. Preparation of LB/AMP/ARA culture medium: 6 1000ml flasks were obtained and 5g of tryptone, 2.5g yeast extract, and 5g NaCl was added to each flash, added DI H2O to a volume of 250ml, shake until solutes dissolve and then bring final volume to 500ml. Adjust the pH to 7.0 with 5M NaOH. Autoclave and let then medium cool to about 40°C and add 1.36g of L-arabinose, and 25mg Ampicillin, shake to dissolve and let it cool down a little more. Now these flasks are ready for inoculation with the culture that has been incubating in the floor shaker. Pour the contents of the 15ml centrifuge tubes into the 6 flasks. Incubate in the floor shaker with vigorous shaking, about 250rpms at 32°C for 16 hours. Remove the flasks from the shaker and examine under long wave UV, the broth should glow bright green. The