A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

用Bac-to-Bac杆状病毒表达系统高效表达绿色荧光蛋白标记的HBVe抗原

通过基因工程操作,使乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）基因与绿色荧光蛋白基因（ＧＦＰ）融合,用新型Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达系统在昆虫细胞中高效表达了ＨＢｅＡｇ－ＧＦＰ双功能融合蛋白。经ＥＬＩＳＡ法和荧光显微镜观察证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＢＶ的ｅ抗原活性,为免疫诊断新方法的建立进行了有益的探索     

Reporter system for the detection of in vivo gene conversion

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr⁶⁶-His⁶⁶). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.     

In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques

An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.     

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.