Scientific Investigation - Lab Report Example

Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling The article en d “Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling” by Andreas Crameri, Erik Whitehorn, Emily Tate, and Willem Stemmer proposes DNA shuffling to acquire a stronger whole cell fluorescence signal for eukaryotic organisms. The research study’s main hypothesis is that a combination of DNA shuffling and either selection or screening would be able to optimize combining positive mutations and simultaneously removing negative mutations from the sequence pool, thus improving fluorescence.

In order to test this hypothesis, an experiment protocol consisting of a number of steps was used. The steps in the entire experiment protocol include (1) GFP gene construction; (2) gene shuffling and selection; (3) mutant selection; (4) mutant characterization in E. coli; (5) CH) cell expression of GFP; (6) FACS analysis; and, (7) fluorescence spectroscopy. That is, the researchers first synthesized a GFP (green fluorescent protein) gene in an E. coli construct with optimized codon usage. This was further improved using an evolutionary process of recursive cycles of DNA shuffling of the GFP gene, combined with visual selection for the brightest E.

Coli colonies. Since red-shifting the excitation maximum makes detection challenging by the naked eye, UV light was used for visual screening. The independent variable in this study is the process used to improve cell fluorescence or GFP in E. coli cells. In particular, the independent variable considered the absence of GFP, the Clontech GFP, the wildtype GFP, the cycle 2 mutant GFP, and the cycle 3 mutant GFP. On the other hand, the dependent variables that were investigated for differences in this study are the fluorescence signals and excitation signals of the E.

coli genes.To control variability, all the constructs were assayed at equal OD. In addition, except for the GFP construct which was induced with IPTG, the other three constructs were induced with arabinose.Technically, the control group for this study is the sample with no GFP. However, the three constructs (i.e., wildtype, cycle-2 and cycle-3) were also compared against the pGFP plasmid from Clontech, with a GFP sequence reported by Chalfie et al and contains a Q80R mutation which occurred as a PCR error as well as 24 extra amino acids from the N-terminus of LacZ.

Results of the research study confirms the initial hypothesis that the combination of DNA shuffling and high throughput screening would be a powerful tool in optimizing a significant number of commercially important enzymes for which selections do not exist.Reference:Crameri, A., Whitehorn, E., Tate, E., & Stemmer, W. (1995). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology , 14, 315-319.