| Abstract [eng] |
To improve heterologous protein expression and growth of laboratory strains of Escherichia Coli, creation of minimal genome strains is becoming more widespread. These minimal genome strains often exhibit rapid growth and grow to higher cell densities. To create these minimal genome strains it is essential to know the loci which can be removed from E. coli genome without consequences to cell growth under laboratory conditions. Until now this essential gene research was focused on single genes only. However, using single gene mutants can lead to misclassifying genes as non-essential because of synthetic lethality. The goal of this work was to analyze whether the genome of E. coli is tolerant to multiple transposon insertions. During the study, a novel system of oscillating plasmids was created. Using this system, which allows to insert multiple Tn5 transposon copies to the genome, an insertional mutant library was generated. 54 of the 96 mutants had at least two insertions in their genome. This proves, that using the aforementioned plasmid system it is possible to create insertional mutant libraries, that have multiple insertions in their genome. |
