Abstract The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing shuffling chromosome parts Cre-loxP system thereby enabling evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in Saccharomyces cerevisiae . L-SCRaMbLE allows tight regulation activity with up 179-fold induction upon exposure red light. extent depends on time...

One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, it generated electrochemical reduction of CO2 and fed to microorganisms soluble form. We previously identified the synthetic reductive glycine pathway most efficient route for aerobic on formate. further demonstrated activity Escherichia coli after expression both native foreign genes. Here, we...

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored one or multiple genes often needed. As TFs from evolutionary distant organisms are unlikely to affect host choice, they represent excellent candidates establishing orthogonal control systems. To establish regulators use yeast (Saccharomyces cerevisiae), we chose the plant Arabidopsis thaliana. We established library 106 different combinations chromosomally integrated...

Orthogonal systems for heterologous protein expression as well the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed characterized a set synTFs based either activator-like effectors or CRISPR/Cas9, small promoters (synPs) with minimal sequence identity to host's endogenous promoters. The resulting collection functional synTF/synP pairs confers...

Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated system use in Saccharomyces cerevisiae. PhiReX is based on the combination customizable synTALE DNA-binding domain, VP64 activation domain light-sensitive dimerization photoreceptor PhyB its interacting partner PIF3 from Arabidopsis thaliana....

Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled generation. It also appears be suitable approach improve ethanol by Saccharomyces cerevisiae. In present study, we constructed different S. cerevisiae strains with heterologous expression genes ATP-hydrolyzing F1-part ATPase enzyme induce enforced and quantify resulting effect on biomass formation.In contrast genomic integration, found...

The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field synthetic biology. Here, we present AssemblX, novel, user-friendly and highly efficient multi-gene strategy. software-assisted AssemblX process allows even unexperienced users to rapidly design, build test with currently up 25 functional units, from 75 or more subunits. At gene level, uses scar-free, overlap-based sequence-independent methods, allowing unrestricted design transcriptional...

Cloning multiple DNA fragments for delivery of several genes interest into the plant genome is one main technological challenges in synthetic biology. Despite modular assembly methods developed recent years, biotechnology community has not widely adopted them yet, probably due to lack appropriate vectors and software tools. Here we present Plant X-tender, an extension highly efficient, scar-free sequence-independent multigene strategy AssemblX, based on overlap-depended cloning rare-cutting...

Abstract A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables assembly of large DNA constructs. The genes cloned into vector were (a) Δ 5 ‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata , (b) steroid ‐isomerase Comamonas testosteronii (c) mutated steroid‐5β‐reductase Arabidopsis thaliana, and (d) 21‐hydroxylase Mus musculus . second bearing an ADR/ADX fusion Bos taurus also...

Viral proteins are highly antigenic and known as potent stimulators of adaptive immune responses. This mechanism is often used for biotechnological applications in monoclonal antibody production resulting high-affinity IgG antibodies most cases. The aim this study was to increase antigen-specific IgA levels mice order generate by hybridoma technology. For purpose, hamster polyomavirus (HaPyV) major capsid protein VP1 immunize different routes induce VP1-specific titers. Recombinant HaPyV-VP1...

Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis reproductive isolation. Apart from molecules such as bindin sea urchins or lysin abalone species, acrosomal protein M7 Mytilus edulis has analyzed. was found be under positive selection, but mechanisms driving evolution this are not fully understood. To explore functional aspects, study investigated expression pattern and M6 in gametes somatic tissue male female M. edulis. The...

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research diagnostic applications. Nevertheless, the specific targeting of complex antigens such whole microorganisms remains a challenge. To provide comprehensive workflow, we combined bioinformatic analyses with novel immunization selection tools to design monoclonal microorganisms. In our initial study, human pathogenic strain E. coli O157:H7 model target identified 53 potential protein...

The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its integrity, despite hybridizing in zones contact with closely related M. trossulus or galloprovincialis. This uses selective antibody production by means hybridoma technology identify molecules which are involved sperm function edulis. Fragmented were injected into mice and 25 cell clones established obtain monoclonal antibodies (mAb). Five identified...

The monoclonal antibody B13‐DE1 binds fluorescein, several fluorescein derivatives, and three peptide mimotopes. Our results revealed that this also catalyzed the redox reaction of resazurin to resorufin, which are both structurally related fluorescein. By using sodium sulfite as a reducing agent, lowered activation energy reaction. Michaelis–Menten constant turnover number were determined 4.2 µmol/l 0.0056 s −1 , respectively. Because showed inhibited catalytic activity antibody, we assume...

Abstract Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage and ribosome or cellsurface formats. We describe a new method which allows the selection Escherichia coli cells producing required single chain cultivation in presence ampicillin conjugated to antigen interest. The utilizes neutralization conjugate produced secreted periplasm. Therefore, expression system based on pET26b vector was designed library...