RESUMEN

Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, are important industrial bacteria. However, there is a lack of standardized and predictable genetic tools for convenient and reproducible assembly of genetic modules in Bacillus species to realize their full potential. In this study, we constructed a Ribosome Binding Site (RBS) library in B. licheniformis, which provides incremental regulation of expression levels over a 104-fold range. Additionally, we developed a model to quantify the resulting translation rates. We successfully demonstrated the robust expression of various target genes using the RBS library and showed that the model accurately predicts the translation rates of arbitrary coding genes. Importantly, we also extended the use of the RBS library and prediction model to B. subtilis, B. thuringiensis, and B. amyloliquefacie. The versatility of the RBS library and its prediction model enables quantification of biological behavior, facilitating reliable forward engineering of gene expression.

Asunto(s)

Bacillus , Bacillus/genética , Bacillus subtilis/genética , Ribosomas/genética , Sitios de Unión , Expresión Génica

RESUMEN

The 5'-untranslated region (5'-UTR) of prokaryotic mRNAs plays an essential role in post-transcriptional regulation. Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, have gained considerable attention as microbial cell factories for the production of various valuable chemicals and industrial proteins. In this work, we developed a portable 5'-UTR sequence for enhanced protein output in the industrial strain B. licheniformis DW2. This sequence contains only ∼30 nt and forms a hairpin structure located right before the open reading frame. The optimized Shine-Dalgarno (SD) sequence was presented as a single strand on the loop of the hairpin for better ribosome recognition and recruitment. By optimizing the free energy of folding, this 5'-element could effectively enhance the expression of eGFP by ∼50-fold and showed good adaptability for other target proteins, including RFP, nattokinase, and keratinase. This 5'-UTR could promote the accessibility of both the SD sequence and start codon, leading to improved efficiency of translation initiation. Furthermore, the hairpin structure protected mRNA against 5'-exonucleases, resulting in enhanced mRNA stability. It is well-known that the stable structure at a ribosome binding site (RBS) impedes initiation in Escherichia coli. In this study, we presented a unique structure at a RBS that can effectively enhance protein production, which is an exception of this prevailing concept. By adjusting a single thermodynamic parameter and holding the other factors affecting protein output constant, a series of 5'-UTR elements with different expression strengths could be rationally designed for wide use in Bacillus sp.

Asunto(s)

Regiones no Traducidas 5'/genética , Bacillus licheniformis/genética , ARN Mensajero/química , Secuencia de Bases , Sitios de Unión , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/metabolismo , Ribosomas/química , Ribosomas/metabolismo

RESUMEN

To evaluate the performance degradation of fabric drying in domestic air-vented dryer, the appearance, mechanical, and microscopic properties of the wool fabric after different drying cycles were studied. Pilling and dimensional stability tests were performed according to established international standards. Microscopic studies were carried out by Scanning Electron Microscopy (SEM) and X-ray (XRD). The results show that pilling is the dominant damage mechanism and gradually increased during lower number of drying cycles (0-10 cycles). The results of dimensional stability showed that the highest dimensional shrinkage of knit wool fabric was first five drying cycles, and then increased at a relatively slow rate with the increase in drying cycles. And fabric shrinkage takes place mainly in the warp direction. The SEM images of wool fiber showed that the scales on the fiber surface were intact in the first 15 drying cycles but start to flake after 20 drying cycles. In the drying process, fabrics can suffer several fractures such as cut fracture, split fracture, partial fracture and distortion and even scale flaking and interior structural distortion of fiber. The damages occur quite randomly, but complexity and severity of damages increased with increase in drying cycles. XRD analysis showed that drying had little effect on fiber crystallinity, indicating that drying only leads to changes in the physical properties of wool fabrics instead of changes in the chemical composition of the fiber. And found SEM analysis is not only an effective method to characterize the change in the surface morphology of the fibers resulting from the drying treatments, but also explain the cause of fabric performance degradation such as pilling and strength reduction during drying.