An automated DIY framework for experimental evolution of Pseudomonas putida

Adaptive laboratory evolution is a general and effective strategy for optimizing the design of engineered genetic circuits and upgrading metabolic phenotypes. However, the specific characteristics of each microorganism typically require the use of exclusive conditions that must be adjusted to the biological chassis at stake. In this work, a do‐it‐yourself (DIY) approach has been used … Leer más

A standardized inverter package borne by broad host range plasmids for genetic circuit design in Gram-negative bacteria

Genetically encoded logic gates (and, in particular, inverters or NOT gates) are the main building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. Nevertheless, the repertoire of inverters readily available for different species is limited. In this work, a variety of NOT gates were recreated as broad host range constructs assembled … Leer más

Capturing Multicellular System Designs Using Synthetic Biology Open Language (SBOL)

Synthetic biology aims to develop novel biological systems and increase their reproducibility using engineering principles such as standardisation and modularisation. It is important to represent and share these systems in a standard way to ensure that they can be easily understood, reproduced, and used by other researchers. The Synthetic Biology Open Language (SBOL) is a … Leer más

For the sake of the Bioeconomy: define what a Synthetic Biology Chassis is!

The role of synthetic biology as a fuel for the bioeconomy requires clarification of the terms typically adopted by this growing scientific-technical field. In particular, the concept of ‘chassis’ as a defined, reusable biological frame where non-native components can be plugged in and out to create new functionalities lies at the boundary between frontline bioengineering … Leer más

Naked Bacterium: Emerging Properties of a Surfome-Streamlined Pseudomonas putida Strain

Environmental bacteria are usually equipped with native surface-attachment elements that can hinder the engineering of biofilms. In this work, the genome of Pseudomonas putida KT2440 was engineered to strip the cells of most of their outer-facing structures, involved in motion, binding to surfaces and biofilm formation. In particular, 23 segments were deleted, resulting in a … Leer más

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E . coli involves 3 proteins (encoded by mutS , mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL ) … Leer más

Digitalizing heterologous gene expression in Gram‐negative bacteria with a portable ON/OFF module

While prokaryotic promoters controlled by signal‐responding regulators typically display a range of input/output ratios when exposed to cognate inducers, non-naturally occurring cases are known to have an OFF state of zero transcription (as ideally needed for synthetic circuits). To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the … Leer más

Multiple-site diversification of regulatory sequences enables interspecies operability of genetic devices

The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts were maintained but their expression levels and stoichiometry diversified by (i) reassembling them together in a single broad host range, standardized vector and (ii) subjecting … Leer más

Pseudomonas putida in the quest of programmable chemistry

Traditional microbial biotechnology is in the midst of a deep transformation brought about by conceptual and technical breakthroughs, as well as by the major change of socioeconomic context generically known as the 4th Industrial Revolution. Pseudomonas putida has recently received a considerable attention as the basis for developing whole-cell catalysts, due to several reasons such … Leer más

Recombination-independent genome editing through CRISPR/Cas9-enhanced TargeTron delivery

Group II introns are retroelements (elements that are transcribed into RNA, reverse-transcribed into DNA and then inserted into a new site in the genome) that are found in bacterial and organelle genomes and that, when in RNA form, are folded into conserved secondary structures. These secondary structures contain six domains, five of which are very … Leer más