Facile preparation of recombinant spider eggcase silk spheres via an HFIP-on-Oil approach.

Versatile spider silk proteins have been prepared by various methods in morphology of spheres for functional applications. Inspired from natural spinning process, a facile approach for the fabrication of silk spheres is described. Distinct from the traditional emulsification method, silk spheres were assembled as rapidly as 10 s by using the HFIP-on-Oil method without any surfactants and agitation used. Notably, a series of factors, such as evaporation rate of HFIP, polarity and molecular weight of oils play central roles on the final silk morphologies. With regard to the increase of protein concentrations, the average dimension and size distribution of silk spheres were both increased. Together with present study, silk spheres prepared by other methods were summarized for comparison in drug delivery applications. As a proof-of-concept, silk spheres loaded with Rhodamine B and Doxorubicin were investigated for the potential proteinase-enhanced drug delivery. On the extracellular environment, ethanol-mediated silk spheres exhibited higher resistance against enzymatic degradation of proteinase K when compared with pristine spheres. Under fluorescent detection by the aid of CLSM, proteinase-enhanced release behaviors were further demonstrated through in-vitro experiments within Hela cells. The facile fabrication of spheres with tunable ß-sheets establishes a fascinating platform for functional silk-based applications.

One-step DNA fragment assembly and circularization for gene cloning.

This article describes a one-step procedure based on Taq polymerase for the precise assembly of DNA fragments into circular constructs as long as 6 kb. The only prior step needed was the amplification of the gene to be cloned and the linear vector backbone, and the whole process up to assembly and circularization lasted only 2 days, compared with the conventional method's 2 weeks. Furthermore, the final DNA construct was used to transform Escherichia coli directly without any further treatment. By circumventing the need for DNA ligase, our "Quick Assemble" method offers an improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-directed mutagenesis and whole-DNA library gene shuffling, as well as the construction of new plasmids with any promoter, resistance gene marker, restriction site, or any DNA tag.