Impact of CRISPR interference on strain development in biotechnology

In this study, straight composite steel-concrete beams were tested to investigate their mechanical performance under combined negative bending and torsional moments. Two specimens were used in this study, and different ratios between the applied negative bending and torsional moments were induced. Load and deflection relationships, strain development on the steel main girder and shear connectors (stud), and the slip development on the steel-concrete interface were recorded in the test and reported in this paper. The results indicate that increase of torsional moment will result in the significant decrease of the load-carrying capacities (e.g. yield load and ultimate load) of the specimens. It was also found that the normal strains of stud shear connectors in such beams are very large and non-negligible compared to their shear strains. In addition, the maximum interface slip was found occurring at around the 1/4 span, and the support conditions and serious crack of the concrete were considered to be the main causes. The research results obtained in this study can provide references for the design and analysis of steel-concrete composite beams subjected to the combined negative bending and torsional moments.

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A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

Communications Biology ◽

10.1038/s42003-021-01672-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Nicole A. Ellis ◽

Byoungkwan Kim ◽

Jessica Tung ◽

Matthias P. Machner

Keyword(s):

Legionella Pneumophila ◽

Bacterial Species ◽

Virulence Gene ◽

Polymerase Activity ◽

Growth Defect ◽

Great Promise ◽

Macrophage Infection ◽

Crispr Interference ◽

Crispr Array ◽

Virulence Protein

AbstractCatalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

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Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin

Molecules ◽

10.3390/molecules26144343 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4343

Author(s):

Lena Mitousis ◽

Hannes Maier ◽

Luka Martinovic ◽

Andreas Kulik ◽

Sigrid Stockert ◽

...

Keyword(s):

Aminoglycoside Antibiotic ◽

Biosynthetic Gene Cluster ◽

Genetically Engineered ◽

Sustainable Production ◽

Biosynthetic Gene ◽

Strain Development ◽

Antibiotic Agent ◽

Phenotypic Stability ◽

High Concentrations ◽

Hydrolysis Of

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound’s purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

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