Erick’s Weblog

ABSTRACT Synthetic life is a form of biological engineering, since specific DNA components are introduced into an organism’s genome. The modified organism will respond as it is programmed. Green Fluorescent Protein (GFP) produces light by fluorescence that is emitted from the Ser-65, Tyr-66, and Gly-67 amino acid sequence. We purified the pGLO plasmid containing GFP using the Alkaline Lysis Plasmid Purification method, verified the GFP gene by restriction digestion of pGLO plasmid using the endonucleases BamHI and HindIII, and visualized the results using agarose gel electrophoresis. We used Bioinformatics techniques and sequence databases to design primer sequences to amplify the GFP gene using the polymerase chain reaction (PCR). We performed PCR and multiple cycles of Error Prone PCR to create GFP mutagenized variants. Then we purified the obtained PCR fragments. In order to verify that the purification was successful we analyzed the results using agarose gel electrophoresis. Then, we ligated the purified PCR fragments to the cloning vector pGEM T-Easy. The vector was trasnformed into E. coli JM109. After that, we purified plasmid DNA from GFP clones and used enzymatic digestion with EcoRI to cut the GFP fragment for DNA sequencing. Our pGLO plasmid purification showed the presence of super coiled plasmid DNA, which confirmed that the purification was successful. We obtained three DNA fragments after restriction digestion of the pGLO plasmid with the enzyme BamHI, and two with HindIII confirming its identity as the pGLO plasmid. Upstream and downstream primers were designed using the Primer 3 Internet program based on the downloaded pGLO gene encoding the GFP protein. PCR reactions amplified a putative GFP fragment of the expected size of approximately 840 base pairs. The ligation was successful because when we transformed E. coli some white ampicilin-resistant fluorescent colonies were obtained. Plasmid DNA from 12 positive colonies were purified and digested with EcoRI liberating DNA fragments identical in size to the PCR products in 5 of the clones as determined by agarose gel electrophoresis. The five digested vector fragments will be sequenced. In the future the variant GFP DNA will be inserted in Halobacterium using the shuttle vector pNG168 in order to characterize the properties of the mutated products.