E. Coli Transformation Lab Report

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The purpose of the lab was to transform E.coli using the plasmid pRFP to promote the expression of antibiotic resistance as well as expression of the red fluorescent protein (RFP). The hypothesis was that if the transformation was successful, then the bacteria would express RFP because the arabinose would activate the plasmid’s red fluorescent protein, and show growth because pRFP allows E.coli to grow even in the presence of an antibiotic. The plasmid was combined with a sample of E.coli through the process of transformation. Following transformation, the protein was isolated using hydrophobic interaction chromatography (HIC) and the size was determined using SDS-PAGE. The results showed that the E.coli transformed by the pRFP would thrive and express the RFP in the proper environment. Through the use of HIC and SDS-PAGE the size of the plasmid was found to be ~27 kDa. Introduction
The purpose of this lab series was to transform E.coli using pRFP and examine the …show more content…

A lysozyme was used to break down walls of the bacteria to allow the RFP to be released. It was hypothesized that after the solution was put through the HIC column, the RFP would be isolated from the bacteria. The HIC matrix binds to the hydrophobic molecules of the protein in high salt conditions. Red fluorescent protein is hydrophobic while bacterial proteins are not. The bacterial proteins will pass through the column while RFP will stay in the column. The first solution that was added, the supernatant, had a high salt content which made the protein bind to the matrix in the column. The second solution, the wash buffer had a lower salt concentration which helped to keep the protein in the the matrix but allowed it to move down and the solution dripped out. The last solution, TE buffer contained no salt which finally led the protein to drip out into the last collection

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