Pglo Transformation Lab Report

Introduction Transformation is a genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surrounding through the cell membrane. The arabinose operon changes AraC from a repressor to an activator; in this experiment the pGLO plasmid has been designed with a modified operon so that in the presence of the arabinose the bacterial cells which have been transformed by the pGLO plasmid will fluoresce due to the production of GFP. SDS-PAGE is a standard technique for determining the abundance and molecular weight of a protein using an anionic detergent (sodium dodecyl sulfate or SDS) which gives the molecules a net negative charge. The charged molecules are pipetted into a gel and then
1ml of 1% arabinose was added to plates 2 and 4, it was spread around and the plates sat and recovered at 37°C. After a few minutes the excess arabinose solution was removed. 100μl of the “+” transformation was pipetted onto plates 1 and 2 while 100μl of “-“ transformation was added to plates 3 and 4, the transformation mixture was then spread around using plating beads, and plates were then left to incubate at 37°C overnight after which they were moved to 4°C for a week. Cell lysates were prepared using B-PER cell lysis reagent and prepared cell cultures, pGLO/DHα + Arabinose, pGLO/DHα – Arabinose, and DH5α – Arabinose. 1.5 ml of each cell culture was added to four separate tubes and each tube was centrifuged at high speed. The tubes were weighed before and after the mixture was added and then the amount of B-PER cell lysis was determined using the weight of the pellet. The pellets were then suspended in B-cell lysis and incubated for 15 min at room temperature. The concentration of cell lysate was determined using a Bradford assay and the samples were diluted accordingly. The Bradford assay was obtained by shining a light with a wavelength of 405 nm through the cell lysate sampled. The data obtained was absorbance and was then plotted vs. the concentrations of the samples. The line
coli using the pGLO plasmid in the presence of different substances affected if the bacteria grew as well as the bacteria’s ability to glow under UV light. Plates 1 and 2 had the pGLO plasmid, plates 3 and 4 did not. The pGLO plasmid used in this experiment had an ampicillin resistant gene, the bla gene, therefore the E. coli which incorporated the pGLO created colonies resistance to ampicillin. The plasmid also had the gene for Green fluorescent protein (GFP) whose expression is controlled by a special regulation system and depends on the presence of ampicillin. Plates 1 and 2 developed the ampicillin resistant colonies. Plate 3 did not have the pGLO plasmid therefore it was not ampicillin resistant and no growth was observed on the plate. Plate 4 did not have pGLO but was not in the presence of ampicillin therefore the growth of bacteria occurred over the whole surface of the plate instead of in colonies. Out of the plates that received the plasmid only two had growth and only one of those two glowed under UV light. Plate 2 is the only plate that glowed, this was due to the presence of arabinose. The arabinose operon mentioned in the introduction is the special regulation system that the expression of GFP was paired. Due to this pairing, only in the presence of arabinose will the GFP gene take part in the transformation and make the colonies glow. This is why only plate 1 has glowing colonies. Sources of error in this lab include malfunctioning