Autores UPV
Pinto Filipe,
Pacheco Catarina C,
Paulo Oliveira,
ARNAU MONTAGUD,
Landels Andrew,
Narciso Couto,
Wright Phillip C,
Urchueguía Schölzel Javier Fermín,
Paula Tamagnini
Abstract
The use of microorganisms as cell factories frequently requires extensive molecular manipulation.
Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required
to ensure a successful outcome. Herewe describe the genome mapping and validation of five
neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium
as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants
were produced, and to assess their functionality, a synthetic green fluorescent reporter module
was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning
site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology
approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic
modules/circuits are also available. This work presents a systematic approach to map and validate
chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.