Improving a Synechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites.
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. Here we 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.
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto 4150-180, Portugal Faculdade de Ciências, Departamento de Biologia, Universidade do Porto, Porto 4150-171, Portugal.
Filipe
F
Pinto
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto 4150-180, Portugal.
Catarina C
CC
Pacheco
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto 4150-180, Portugal.
Paulo
P
Oliveira
Instituto Universitario de Matemática Pura y Aplicada, Universitat Politècnica de València, Valencia 46022, Spain.
Arnau
A
Montagud
ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S10 2TN, UK.
Andrew
A
Landels
ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S10 2TN, UK.
Narciso
N
Couto
ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S10 2TN, UK.
Phillip C
PC
Wright
Instituto Universitario de Matemática Pura y Aplicada, Universitat Politècnica de València, Valencia 46022, Spain.
Javier F
JF
Urchueguía
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto 4150-180, Portugal Faculdade de Ciências, Departamento de Biologia, Universidade do Porto, Porto 4150-171, Portugal pmtamagn@ibmc.up.pt.
Paula
P
Tamagnini
D052818:Autotrophic Processes; D002874:Chromosome Mapping; D000458:Cyanobacteria / Q000235:genetics* / Q000254:growth & development; D049452:Green Fluorescent Proteins; D009154:Mutation; D046939:Synechocystis / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods
D049452:Green Fluorescent Proteins
Synechocystis; neutral sites; photoautotrophic chassis; synthetic biology