Construction of CRISPR Libraries for Functional Screening.

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

A Rapid Combinatorial Approach to Assembling Synthetic Prokaryotic and Eukaryotic Protein Expression Vectors.

Vector construction and gene cloning are ubiquitous techniques essential to all fields of biological and medical research. They are the first steps in many endeavors leading to expressing proteins to understand gene function and regulation. However, they can often be rate-limiting, particularly in multi-gene studies, due to the time and effort required to assemble gene constructs and to identify the optimal constructs for protein expression.The SureVector system was developed to address this by enabling the rapid and reliable assembly of multiple DNA modules into a recombinant plasmid containing a gene-of-interest (GOI). It harnesses the power of synthetic biology to combine DNA modules from standard parts into a customized vector that expresses proteins in bacterial, mammalian, or yeast cells. The key advantages of the innovative SureVector system include rapid custom vector generation, enhanced flexibility to assemble new vectors quickly as experimental requirements change, and the reliable and precise assembly of fully interchangeable standard DNA modules that retain their functionality. The SureVector system is the only next-generation plasmid assembly technology to guarantee assembly of multiple functional DNA modules.

Impact of wine manufacturing practice on the occurrence of fining agents with allergenic potential.

Proteinogenic wine fining agents are hidden allergens and could present a risk for consumers with allergies. Therefore, the European Parliament adopted Directive 2003/89/EC amending Directive 2000/13/EC to declare ingredients, contaminations and processing aids that are known to trigger allergic reactions. The Amendment Regulation (EU) 1266/2010 excluded the labelling of wines which are processed with hen's egg and products thereof until 30 June 2012 to get more scientific findings. After 1 July 2012 wine fining agents have to be declared if above 0.25 mg l(-1) (Regulation (EU) 579/2012 in conjunction with article 120 g of Regulation (EU) 1234/2007). The Organisation International de la Vigne et du Vin (OIV) advises this limit of detection (LOD) for potential allergenic residues of proteins. Wine fining agents are processing aids and according to the wine producer's knowledge will be removed after coagulation by filtration or other production steps. Due to lack of scientific data, residues of fining agents in the final product could not be excluded. In this risk assessment, highly sensitive ELISA methods for ovalbumin of known origin for wine have been developed. The objective was to investigate the presence of allergen residues in wine after certain technological treatments were applied to remove the wine fining agents. For all developed ELISA methods the LODs are in the low µg l(-1) range between 5 and 10 µg l(-1) fining agent, whereas the LOQ varies between 5 and 80 µg l(-1) fining agent. The results of the investigation of well-known wines and fining agents demonstrate that white wines fined with white or ovalbumin from hen's egg could retain allergens. The use of certain technological procedures during wine processing leads to different results. In white wine, bentonite or sheet filtration followed by sterile filtration lead to wines containing no detectable amounts of ovalbumin. In red wine, especially the final sterile filtration removes the fining agents.

Evaluation of the efficiency of enological procedures on lysozyme depletion in wine by an indirect ELISA method.

Potential residues of the potent allergen lysozyme used as a microbial stabilizing agent in wine production might pose a serious health thread to susceptible individuals. Therefore, EU legislation requires the labeling of the allergenic agent, if it is present in the final product. To allow for product testing, an indirect ELISA method to be specifically used in wine analysis was developed and validated. Furthermore, trial wines treated with defined amounts of lysozyme were subjected to an array of different filtration and other enological processing regimes in order to evaluate their potential to deplete the allergen content of the wines. By these means, processing methods ought to be identified that can be integrated in a good manufacturing practice guideline to enable wine producers to utilize lysozyme in their cellars and still provide wines free of allergenic residues. However, among the enological procedures under scrutiny, only bentonite fining proved to be capable of significantly reducing the allergenic residues.

Development of a sensitive ELISA for the detection of casein-containing fining agents in red and white wines.

Fining of wine with proteinogenic fining agents such as casein from cow's milk is a traditional and commonly used technique all over the world. Casein and other proteins from cow's milk are well-known food allergens, which pose a risk for allergic consumers. Temporary regulations exempting the labeling of milk and products thereof in wine expired. Since July 1, 2012, these fining agents have to be declared on the wine label under Regulation (EU) No. 579/2012 in conjunction to article 120g of Regulation (EU) No. 1234/2007 if exceeding the threshold of 0.25 mg/L allergenic protein. The aim of the presented study was to develop sensitive ELISA methods for the detection of casein in white and red wines and to investigate the risk of allergenic residues in fined wines. In this context it was shown that the used substance for calibration is highly relevant. Casein wine fining agents of different commercial producers were investigated by LDS-PAGE and immunoblot. In addition to casein, they contain other milk proteins, which are potentially allergic and therefore have to be incorporated in the development of antibodies for an ELISA method to be set up. An indirect ELISA for the investigation of white wine was developed. The LOD is 0.1 mg/L. For red wine the LOD is 0.2 mg/L in an indirect sandwich ELISA setup. The LOD of the indirect sandwich ELISA for white wine depends on the calibration standard. It is 0.1 mg/L for the fining agent casein and 0.01 mg/L for casein from a chemical trader. It is also shown that the use of different technological procedures during winemaking leads to no detectable amounts of casein in various wine samples.