Design and Assembly of DNA Sequence Libraries for Chromosomal Insertion in Bacteria Based on a Set of Modified MoClo Vectors.
Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most popular assembly systems is the MoClo standard in which restriction and ligation of multiple fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs restriction enzymes, which cut outside their recognition site. While the initial MoClo vectors had been developed for the assembly of multiple transcription units of plants, some derivatives of the vectors have been developed over the last years. Here we present a new set of MoClo vectors for the assembly of fragment libraries and insertion of constructs into bacterial chromosomes. The vectors are accompanied by a computer program that generates a degenerate synthetic DNA sequence that excludes "forbidden" DNA motifs. We demonstrate the usability of the new approach by construction of a stable fluorescence repressor operator system (FROS).
Chromosome Biology Group, LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-Universität Marburg , Hans-Meerwein-Str. 6, D-35043 Marburg, Germany.
Daniel
D
Schindler
Chromosome Biology Group, LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-Universität Marburg , Hans-Meerwein-Str. 6, D-35043 Marburg, Germany.
Sarah
S
Milbredt
Chromosome Biology Group, LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-Universität Marburg , Hans-Meerwein-Str. 6, D-35043 Marburg, Germany.
Theodor
T
Sperlea
Chromosome Biology Group, LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-Universität Marburg , Hans-Meerwein-Str. 6, D-35043 Marburg, Germany.
Torsten
T
Waldminghaus
D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology
Escherichia coli; chromosome; genome engineering; sequence design; software; synthetic biology