Lab partner- Kim Harcourt
Lab report- Experiment 1: Microbial Genetics
The objective of this experiment was to introduce the study of bacterial genetics in order to identify the potency of different mutagenic agents. Our primary aim was to demonstrate the different techniques needed to isolate biosynthetic auxotrophic mutants using chemical, physical and transposon mutagenesis. The second aim was to plan and execute an experiment designed to isolate catabolic mutants (Lac-) as well as conditional mutants (Temperature sensitive) using only a chemical treatment (EMS). Also, to make and characterize transposon-insertion mutants of KL14 using bacteriophage as a transposon delivery vector. To identify and demonstrate the techniques needed to enrich and isolate the biosynthetic auxotrophic, catabolic auxotrophic and conditional mutants. Lastly, we were to characterize biosynthetic auxotrophic mutants and effectively characterize itʼs biosynthetic pathway. 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). The TS phenotype is thought to have arisen from unfolding and consequent inactivation of the mutant polypeptide chain at the restrictive temperature. The catabolic mutant that we worked with was the lactose negative mutant. Lactose metabolism requires two speciﬁc proteins, a permease (product of the lacY gene) and has the role of transporting lactose into the cell. The second protein is the ϐ-galactosidase (product of lacZ gene) an enzyme that hydrolyses lactose to yield glucose and galactose. A cell which fails to synthesize either of these proteins, or in which one of the proteins is defective, it will be unable to use lactose. The shorthand notation for this is lac-, the defective genes can either be speciﬁed as lacZ- or lacY. Various mutagenic treatments were used in the form of chemical, physical and genetic mutagenesis in order to induce and isolate the auxotrophic and conditional mutants. The chemical agent that we used was Ethylmethanesulphate (EMS), which is an alkylating agent that induces chemical modiﬁcation of nucleotides, resulting in mispairing and base changes. EMS produces random point mutations in genetic material by nucleotide substitution, typically inducing C to T changes, which results in C/G to T/A substitutions. EMS also produces transversions as well as small deletions. Ultraviolet radiation was used as a physical agent to induce both base substitutions and deletions in the E.coli cell culture. Exposure to the UV light causes dimers to form between adjacent pyrimidine residues, or between pyrimidines of complementary strands which interferes with normal base pairing. The C---> T transition is also the most frequent mutation observed but other transversions, frameshifts and deletions are also stimulated by UV light. The third type of treatment used was transposon...