Biosynthetic Auxotroph Lab Report

Register to read the introduction… We determined that the UV light treatment was the most potent treatment of mutagenesis and also the most effective in inducing mutations, followed by the Tn5 and the EMS was the least potent and effective treatment.

A culture of E.coli K12, KL14 was used to carry out the experiment on. The advantage of using E.coli cells to experiment on is that they are haploid organisms and contain a single set of chromosomes so if a mutation was to occur, the bacterial cell will express it as there is only one copy of the gene. We experimented on various types of mutations. Auxotrophic mutants are impaired in their metabolic capabilities, they can either be biosynthetic auxotrophs which require an additional nutrient in the minimal media in order to grow or can be catabolic auxotrophs, which are affected in their ability to utilize a particular substance for growth. The phenotype of a conditional mutant is expressed only when the organism is grown under a particular set of conditions. Temperature sensitive (TS) strains grow normally at the permissive temperature (30°C-33°C) but do not grow at a restrictive temperature (39°C-42°C).
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The EMS treatment was performed until 99.9% of the E.coli cells were killed, as this was the peak time that the ideal amount of mutagenesis was achieved and minimum number of multiple mutations in a cell. We followed the protocol as described in page 14-17 of the lab manual. However even though mutagenesis was achieved through EMS treatment, we would have to screen 100-1000 plates to isolate one mutant, therefore in part II of the experiment penicillin enrichment was used. We calculated that 10 µg of ampicillin would be added to the culture of mutagenised cells. The rest of the experiment was conducted as stated in the lab manual following part III onwards to part VIII, we screened for any biosynthetic auxotrophic mutants, picked cells from the mutant colonies, created pure mutant cultures and finally we identified the specific nutritional requirement of each of the mutants we obtained. When starting pure mutant colonies, we ensured that mutant colonies were firstly streaked onto a minimal medium and then a NA plate, this was so no additional nutrient was transferred onto the selective media when streaking the mutant onto two plates, otherwise the biosynthetic auxotroph would have grown on both plates. The second part of this experiment utilized UV light as the physical mutagenesis treatment on the cell culture. The duration that the cells were exposed for played a key role in determining the 99.9% killing of the cells. From analyzing each serial-dilution of the corresponding time-sample onto NA plates, we were able to identify 99.9% killing as a 1000-fold reduction in the cell population from 0 seconds onwards, by using a semi-quantitative method and counting the colonies that had developed (30 seconds). Using this dilution we continued as stated from part III in the manual. The

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