PubMed · 12824633
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Trevor McFarland, Binoy Appukuttan, J Timothy Stout. 2003. Retinal tissue.. https://doi.org/10.1385/1-59259-393-3%3A205
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The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.
Given a nascent protein sequence, how can one predict its signal peptide or "Zipcode" sequence? This is an important problem for scientists to use signal peptides as a vehicle to find new drugs or to reprogram cells for gene therapy (see, e.g. K.C. Chou, Current Protein and Peptide Science 2002;3:615-22). In this paper, support vector machines (SVMs), a new machine learning method, is applied to approach this problem. The overall rate of correct prediction for 1939 secretary proteins and 1440 nonsecretary proteins was over 91%. It has not escaped our attention that the new method may also serve as a useful tool for further investigating many unclear details regarding the molecular mechanism of the ZIP code protein-sorting system in cells.
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