Search

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Some microbial toxins also target the producer species itself, necessitating a means of self-protection. The M2 double-stranded RNA (dsRNA) killer virus in Saccharomyces cerevisiae contains a single open reading frame (ORF) encoding both the secreted pore-forming toxin K2 as well as a cognate immunity factor. Here, we show that expression of a 49-amino acid N-terminal peptide from the K2 precursor is both necessary and sufficient for immunity. This immunity peptide simultaneously functions as a signal peptide for toxin secretion and protects the cell against the cytotoxic K2 α subunit. The K2 toxin and immunity factor can be functionally separated into two ORFs, yielding a modular toxin-immunity system. This case further shows how a (signal) peptide can carry the potential for providing cellular protection against an antimicrobial toxin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae -based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK ( C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ∼350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Background: Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system., Results: We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations., Conclusion: Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

The oral pathogen Porphyromonas gingivalis is one of the major periodontal agents and it has been recently hailed as a potential cause of the autoimmune disease rheumatoid arthritis. In particular, the peptidylarginine deiminase enzyme of P. gingivalis (PPAD) has been implicated in the citrullination of certain host proteins and the subsequent appearance of antibodies against citrullinated proteins, which might play a role in the etiology of rheumatoid arthritis. The aim of this study was to investigate the extracellular localization of PPAD in a large panel of clinical P. gingivalis isolates. Here we show that all isolates produced PPAD. In most cases PPAD was abundantly present in secreted outer membrane vesicles (OMVs) that are massively produced by P. gingivalis, and to minor extent in a soluble secreted state. Interestingly, a small subset of clinical isolates showed drastically reduced levels of the OMV-bound PPAD and secreted most of this enzyme in the soluble state. The latter phenotype is strictly associated with a lysine residue at position 373 in PPAD, implicating the more common glutamine residue at this position in PPAD association with OMVs. Further, one isolate displayed severely restricted vesiculation. Together, our findings show for the first time that neither the major association of PPAD with vesicles, nor P. gingivalis vesiculation per se, are needed for P. gingivalis interactions with the human host.

The immunodominant staphylococcal antigen A (IsaA) is a potential target for active or passive immunization against the important human pathogen Staphylococcus aureus. Consistent with this view, monoclonal antibodies against IsaA were previously shown to be protective against S. aureus infections in mouse models. Further, patients with the genetic blistering disease epidermolysis bullosa (EB) displayed high IsaA-specific IgG levels that could potentially be protective. Yet, mice actively immunized with IsaA were not protected against S. aureus infection. The present study was aimed at explaining these differences in IsaA-specific immune responses. By epitope mapping, we show that the protective human monoclonal antibody (humAb) 1D9 recognizes a conserved 62-residue N-terminal domain of IsaA. The same region of IsaA is recognized by IgGs in EB patient sera. Further, we show by immunofluorescence microscopy that this N-terminal IsaA domain is exposed on the S. aureus cell surface. In contrast to the humAb 1D9 and IgGs from EB patients, the non-protective IgGs from mice immunized with IsaA were shown to predominantly bind the C-terminal domain of IsaA. Altogether, these observations focus attention on the N-terminal region of IsaA as a potential target for future immunization against S. aureus.

We previously reported that higher serum concentrations of C-reactive protein (CRP) are associated with shorter survival in men with castration-resistant prostate cancer (CRPC). To confirm this finding in an independent data set, we used 119 CRPC patients enrolled in 6 phase II clinical trials and examined the relationship of CRP, alkaline phosphatase, hemoglobin, age, ECOG PS, and prostate specific antigen (PSA) with survival. Median follow-up was 19.7 months (0.9-98.5 months), and 89% have died. After analyzing the form of the risk function using the generalized additive model method, univariate and multivariate Cox proportional hazard models were used to assess associations between baseline individual categorical and continuous variables. Quartiles of CRP were: 0-1.0, 1.1-4.9, 5.0-17.0, and 17.1-311 mg/L. In a Cox multivariate model, log(2) (CRP) (HR 1.106, P = 0.013) as well as hemoglobin and alkaline phosphatase were independently associated with survival, confirming that higher CRP is associated with shorter survival in CRPC. Since CRP is a marker of inflammation, this finding suggests that inflammation may play an important role in the natural history of advanced prostate cancer. CRP is a readily measurable biomarker that has the potential to improve prognostic models and should be validated in a prospective clinical trial., (Copyright © 2012 Elsevier Inc. All rights reserved.)