Synthetic secondary chromosomes in Escherichia coli

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Torsten Waldminghaus, Sonja Messerschmidt, Sarah Milbredt, Daniel Schindler, Nadine Zimmer and Torsten Waldminghaus

SYNMIKRO - LOEWE Center for Synthetic Microbiology, Germany

Advances in de novo synthesis of DNA and assembly methodology make construction of synthetic chromosomes a reachable goal. Considering a potential design leads almost certainly to the question what the essential parts of a chromosome are. Investigations on this question have mainly focused on the minimal set of genes needed to allow cells to live. However, chromosomes are more than arrays of genes. Chromosomes need systems to replicate, segregate and organize the encoded genetic information. We explore such chromosome maintenance systems by application of synthetic biology approaches. Our goal is the design and assembly of secondary synthetic chromosomes in Escherichia coli. The natural template is the human pathogen Vibrio cholerae which carries a natural secondary chromosome. Chromosome maintenance systems usually consist of proteins binding to sites with specific chromosomal distributions. The construction of synthetic secondary chromosomes will allow variation of this binding site distribution. Downstream functional characterization should allow deeper understanding of chromosome maintenance. As a proof of principle we designed a set of three secondary synthetic chromosomes with varied distribution of GATC sequences. GATC is the target sequence for MutH, involved in DNA mismatch repair and SeqA, which is involved in chromosome segregation. The synthetic chromosomes are designed to bind only MutH, the dimeric SeqA and MutH or none of the proteins. Analysis of mutation rates should provide insights into a potential functional interplay of mismatch repair and chromosome segregation. Our long term goal is to establish synthetic secondary chromosomes as experimental system to study chromosome maintenance and to provide chromosome construction rules for biotechnology applications.