Heidelberg 2017

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As fellow scientists exploring the field of targeted evolution, we helped iGEM-team Heidelberg by conducting an experiment for them, to validate their results in another lab. Our colleagues used three different mutagenesis plasmids (MPs) created by A. H. Badran et al. in 2015: #1, #2 and #3, as they are named by the iGEM team. The MPs build up on each other in accession of their respective numbers.

#1 increases the mutation-rate through arabinose induces expression of dnaQ926, an ineffective E. coli DNA Pol III proofreading domain.

#2 also contains the gene encoding Dam methylase under arabinose induction, as well as the gene seqA under low-level expression. Dam methylase is crucial in mismatch-repair in E. coli, as it methylates the parental strand in DNA replication, making it distinguishable from the new daughter strand. If Dam methylase is overexpressed, the daughter strand is methylated too quickly, which impairs the mismatch-repair mechanism. seqA encodes a protein that delays the function of Dam methylase, which solves some issues of the arabinose induced promoter being not completely tight.

#3 contains the additional genes ugi, cda1 and emrR. ugi encodes cytidine deaminase, which catalyzes cytosine to tyrosine transitions and therefore causes base substitutions in the host’s genome. This transition goes through a deoxyuracil intermediate, which may be excised by uracil-DNA glycosylase during the native uracil-excision pathway. cda1 is a natural inhibitor of uracil-DNA glycosylase and therefore acts synergistically with ugi. emrR is a gene repressor for a number of different multidrug resistance pumps. The exact mechanism is unknown, but overexpression of emrR leads to a repression of those efflux pumps and therefore may lead to an increase of mutagenic pathway intermediates in the cell, increasing mutagenesis.

#1, #2 and #3 raise mutagenesis rates high enough, for E. coli to spontaneously evolve antibiotic resistances. If plated on several different antibiotics, under induction of the plasmids with arabinose, a portion of the cell population is supposed to survive with new resistances. This is what Heidelberg wants us to verify. Below is their protocol, which we followed:

Results

Munich

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INSA-UPS France - Toulouse

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