ProjektportalRepeatability in evolution of generalist enzymes
Terrmin
HT23
Beskrivning
In my research group, we use experimental evolution and bacterial genetics to study various aspects of evolution, in particular regarding evolution of new genes. In one of our model systems we use mutants of Salmonella enterica lacking the gene trpF (see Box 1), in order to study how other genes can evolve a new function (replacing the missing enzymatic activity of TrpF). The results of these studies will answer fundamental questions in evolutionary biology, but may also have implications for our understanding of evolution of new antibiotic resistance genes.
In an experiment designed to test a hypothesis about the evolution of bifunctional “generalist” enzymes we have identified multiple mutations in the genes hisA and trpA (see Box 1) that can replace the function of trpF while still maintaining their original functions.
This project is aimed at determining how repeatable evolution is with regards of which mutations are selected in trpA, with the aim of finding out if there are few or many ways of evolving TrpA to become a good generalist (with TrpF and TrpA activity). We will also find out if TrpA is still likely to evolve to become a generalist if it is allowed to lose its original function.
In order to do this, evolution experiments with multiple lineages of strains with either the wild type trpA or some of the previously identified “starting mutations” in trpA will be evolved through serial passage in the same conditions as the original evolution experiment and in slightly different conditions. To speed up evolution the strains will also have a “mutator” mutation (mutS) which increases the mutation rate several fold and speed up evolution.
The evolution experiments will be done by serial passaging of batch cultures in media with a very limited amount of tryptophan, where gain of TrpF activity confers a great fitness advantage. In order to find additional “starting point” mutations we will evolve several populations from a wild-type ancestor, and to see how reproducible evolution from the same “starting mutation” is we will evolve multiple populations from one or two of the already identified “starting mutations”. Finally, in order to determine the likelihood of TrpA evolving to become a generalist enzyme in conditions where it is allowed to lose its original function, we will evolve strains with two copies of TrpA (so that one copy will always keep the original function).
Methods that will be used:
Experimental evolution through serial passage
PCR
DNA sequencing
Competition experiments
Transductions using phage P22
Lambda Red recombineering
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Box 1:
Generalist enzyme – an enzyme that has evolved to catalyze more than one reaction (or act on more than one substrate).
Specialist enzyme – an enzyme that has evolved to catalyze a single reaction (or act on a soingle substrate).
TrpF – PRA isomerase – catalyzes the third step in the synthesis of the amino acid tryptophan. Encoded by the gene trpF in the trp operon. Required for growth in medium lacking tryptophan.
HisA – ProFAR isomerase – catalyzes the fourth step in the synthesis of the amino acid histidine. Encoded by the gene hisA in the his operon. Required for growth in medium lacking histidine.
TrpA – tryptophan synthase ? subunit, together with tryptophan synthase ? subunit (TrpB), catalyzes the final step in the synthesis of the amino acid tryptophan. Encoded by the gene trpA in the trp operon. Required for growth in medium lacking tryptophan.
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Huvudområde
Farmaceutisk vetenskap
Ämne
Mikrobiologi, infektionsbiologi
Typ
Laborativ studie
Företag
Uppsala Universitet
Ort/Plats
Uppsala
Handledarens namn
Joakim Näsvall
Handledarens e-post
joakim.nasvall@imbim.uu.se
Institution
Institutionen för medicinsk biokemi och mikrobiologi
Program
Apotekarprogrammet
Kurs
Fördjupningsprojekt i farmaceutisk mikrobiologi 30 hp - 3FM212
Omfattning/hp
30hp
Hur många studenter kan antagas för detta projekt?
1