The objectives of the experiments performed in the past three weeks were to induce mutagenesis in the E.coli K-12 strain W3110 through short ultra violet light (UV). This particular strain of E.coli lacked photolyase (phr) and the uvr system, which are non-mutagenic repair mechanisms. A survival curve was constructed based off various irradiation times and number of viable cells obtained. Mutant strains were then isolated and purified on MacConkey (MAC) plates to rule out false positives. During week 7, the generation time of the potential Lac+ and lac- strands of E.coli were also determined. The lac operon was also induced via IPTG to determine if β-galactosidase induction is affected by mutations. During week 8, we determined if β-galactosidase …show more content…
After irradiation of the cells, each group examined their cells under phase contrast. As shown in figures 1-7, the cells were rod shaped, some motile, and the rods varied in length depending on exposure time. The cells that were not exposed to UV light (Figures 1-2) appeared like they were thicker than the other cells that were exposed to UV light. Based on the pictures, it is inferred that as the exposure time increased, there were less replicated and dividing cells. This observation is expected when the SOS system is under stress (UV light) because when the system is under stress less cells are dividing, hence smaller amounts of cells as exposure time increase. It appeared that the longer the exposure time, the longer the rods. Some of the pictures taken are inconsistent with this trend because some of the cells are smaller as exposure time increases, while some are larger when exposure time increases. After observing the cells during lab, it appeared that some of the cells lost some of their motility as exposure time increased. Serial dilutions (ranging from 100-10-5) was then performed to make it easier to find mutations in the Lac genes before plating it on MAC plates. MAC plates were used because it can detect whether or not the colony was a mutant. Lac mutants appear pinkish or white on the MAC plates because they cannot ferment lactose. My group (8) irradiated the E.coli for …show more content…
IPTG binds to the repressor stimulating transcription of the lac operon synthesizing β-galactosidase. In E.coli, mutations due to UV exposure can change the enzyme’s function, cause deletions of the Lac Z or Lac Y genes, or even delete the entire operon. All these could prevent β-galactosidase from synthesizing. To determine whether or not β-galactosidase was activated with the addition IPTG, a colorimetric assay was used. The total amount of β-galactosidase produced was indicated by the yellow color resulting in cleavage of the ONPG by β-galactosidase. Our group (Lac mutant) did not observe any yellow coloration after 12 minutes. Miller units were used because it measures the activity of β-galactosidase, and can be calculated using the formula:units=1000 x (OD420-1.75xOD550)/(t x V x OD600). Figure 12 shows the graph and table of Miller units after induction. The graph consists of our Lac mutant (50 microliters) and Nick’s Lac+ from group 4 (50 microliters). The values used to construct the graph were picked based on the sample that fit best within the range of 0-1. The Lac+ strain had positive Miller units that increased over time, indicating that β-galactosidase was synthesized. The Lac mutant remained at 0 over time possibly indicating a lack of β-galactosidase. This could result in a