uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost. Here, we present a scalable, rapid, and inexpensive approach for creating User-designed Physically Isolated Clonal–Mutant (uPIC–M) libraries that utilizes recent advances in oligo synthesis, high-throughput sample preparation, and next-generation sequencing. To demonstrate uPIC–M, we created a scanning mutant library of SpAP, a 541 amino acid alkaline phosphatase, and recovered 94% of desired mutants in a single iteration. uPIC–M uses commonly available equipment and freely downloadable custom software and can produce a 5000 mutant library at 1/3 the cost and 1/5 the time of traditional techniques.

Category-free complement selection in causal adjunct phrases

In English there are a variety of causal adjunct phrases such as because of, as a result of, on account of and in spite of. It was reported recently that a new structure because X is thriving in colloquial registers including conversations and blogs. The complement X is not only restricted to nominals but also includes other lexical categories such as adjectives, adverbs and even verbs. This article delves into the history of this usage and its reasoning, and conducts a survey on other causal adjuncts to determine whether the same kind of innovation is observed with other adjunct phrases. The survey shows that the new usage started from NP complements and has been extended to include complements of other lexical categories, and that the new usage attested in because X is also observed with in case X. The truncation of the final preposition is verified with all the adjunct phrases in the survey, but the category of the complement is basically restricted to nominals in the case of other phrases. We will look into the factors segregating the two groups of adjuncts, namely because/in case X, and other causal adjuncts.

A Cognitive-semantic Study of Verbal Complementation with Adjectives Expressing the Emotion of Fear in Canadian English

This paper looks at the variation in Canadian English between to-infinitive and of + gerund-participle complements with six adjectives that have as part of their semantic makeup a notion of fear. Using data from the Strathy Corpus of Canadian English and the Canadian component of the Global Web-Based Corpus of English, it aims to explain the reasons underlying complement selection with these adjectives as well as temporal relations between the emotion expressed by the adjective and that expressed by the complement. The complementation patterns examined in this study are shown to be connected to certain of the conceptual metaphors that previous studies have observed to be utilized to conceptualize the emotion of fear.