Table of Contents:
Eukaryotes and prokaryotes differ in the organization of their genomes. Most prokaryotes contain a single, circular chromosome that is replicated by one single replication origin. Contrary, eukaryotes possess multiple, linear chromosomes, which are replicated by more than one replication origin. Whether, and to what extent, Escherichia coli tolerates an eukaryotic genome organization is an exciting question whose answer could help to develope construction rules for synthetic chromosomes. This thesis presents DNA replication studies of E. coli strains with multiple-origins on the chromosome and an E. coli strain with two linear chromosomes. Additionally, a fluorescence-based single-cell method has been designed and established, which has an improved "signal to background ratio". The main results can be summarized as follows:
I. E. coli tolerates additional replication origins on the native chromosome, which are able to initiate DNA replication. DNA replication activity of extra copies of oriC or oriII, the replication origin of the second chromosome of V. cholerae, could be verified. A shortened sequence of oriC, which does not contain DnaA box R4, is not sufficient to initiate DNA replication on the native chromosome. If oriII is used as additional replication origin, DNA replication shows dysregulated patterns, which are abolished if oriC and oriII locate on two different replicons within the cell.
II. For design and assembly of DNA sequence libraries for chromosomal integration into bacteria the program MARSeG (Motif Avoiding Randomized Sequence Generator) was written and the modular cloning system MoClo was modified. MARSeG allows the design of sequences with a high variability and the simultaneous exclusion of certain motifs. MoClo has been optimized to increase cloning efficiency, to simplify assembly operations in library scale and to facilitate genomic integration of assembled DNA fragments. MARSeG and the modified MoClo vector were used to design and construct a LacO/TetO hybrid FROS array.
III. A low background FROS (Fluorescence Repressor Operator System) was designed and established. The new method BiFCROS (Bimolecular Fluorescence complementation and Repressor Operator System) is based on fusions of repressor proteins with a split fluorescent protein, which bind to a hybrid FROS array resulting in fluorescence signals due to bimolecular fluorescence complementation. Background fluorescence is reduced, because only proteins that are bound to the hybrid FROS array fluoresce. In order to analyze whether BiFCROS could be used for copy number determination of gene loci, it was further developed in this direction. Thus, total cell fluorescence intensity should reveal the copy number of the hybrid FROS array, because it should increase proportionally with increasing copy number.