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

Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

Pseudomonas putida has many naturally-evolved metabolic and stress-tolerant properties, which makes this organism ideal for many biotechnological applications. Glucose catabolism, a redox reaction that allows to obtain energy from carbohydrates, takes place in P. putida KT2440 through the Entner-Doudoroff (ED) pathway. Through this pathway, glucose is catabolized into pyruvate through a series of enzyme-assisted reactions. At one point, … Leer más

A simple workflow to identify, clone and quantify the function of recombinases in Gram-negative bacteria

The endogenous RecA homology-based recombination system has been typically used to edit the bacterial chromosome in Gram-negative bacteria, and although this technique is efficient, it is also time-consuming. An alternative to this is the use of recombineering, which is based on the use of recombination proteins derived from bacteriophages that stimulate the recombination of incoming … Leer más