[Research on the blended learning mode of "Microbial Breeding Experiments" based on WeChat].

With the rapid development of the internet technology, the blended learning mode plays a more and more important role in education reforms by integrating traditional classroom teaching and online learning. WeChat is the most popular Chinese social software, and its public platform is also suitable for mobile learning. Here, we report an application of the blended learning method based on WeChat public platforms in microbiology breeding experiment courses, using the site-directed mutagenesis of the green fluorescent protein (GFP) as an example. The learning process was divided into five modules: teaching design, learning resource preparation, pre-class learning, classroom learning, post-class review and evaluation. By introducing one mutation (Y66H) in mutagenic primers, the mutated GFP gene was amplified by PCR using pGFPuv as templates, followed by removal of the original plasmid template by Dpn1 digestion. Students can monitor the color changes from green to blue in the fluorescence emission of the mutated proteins. As a useful addition to classroom teaching, WeChat is suitable for students to use fragmented time to learn and improve teaching interaction. Learning assessment results revealed the blended learning environment improves students' study interests and self-learning abilities, thus achieving a fruitful teaching result.

[Comparative genomics and evolutionary analysis of CRISPR loci in acetic acid bacteria].

The clustered regularly interspaced short palindromic repeat (CRISPR) is a widespread adaptive immunity system that exists in most archaea and many bacteria against foreign DNA, such as phages, viruses and plasmids. In general, CRISPR system consists of direct repeat, leader, spacer and CRISPR-associated sequences. Acetic acid bacteria (AAB) play an important role in industrial fermentation of vinegar and bioelectrochemistry. To investigate the polymorphism and evolution pattern of CRISPR loci in acetic acid bacteria, bioinformatic analyses were performed on 48 species from three main genera (Acetobacter, Gluconacetobacter and Gluconobacter) with whole genome sequences available from the NCBI database. The results showed that the CRISPR system existed in 32 species of the 48 strains studied. Most of the CRISPR-Cas system in AAB belonged to type I CRISPR-Cas system (subtype E and C), but type II CRISPR-Cas system which contain cas9 gene was only found in the genus Acetobacter and Gluconacetobacter. The repeat sequences of some CRISPR were highly conserved among species from different genera, and the leader sequences of some CRISPR possessed conservative motif, which was associated with regulated promoters. Moreover, phylogenetic analysis of cas1 demonstrated that they were suitable for classification of species. The conservation of cas1 genes was associated with that of repeat sequences among different strains, suggesting they were subjected to similar functional constraints. Moreover, the number of spacer was positively correlated with the number of prophages and insertion sequences, indicating the acetic acid bacteria were continually invaded by new foreign DNA. The comparative analysis of CRISR loci in acetic acid bacteria provided the basis for investigating the molecular mechanism of different acetic acid tolerance and genome stability in acetic acid bacteria.

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin.

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by (60)Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.