A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.     

The sequence upstream of the −10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli

We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known −10 consensus sequence, as well as the extended −10 region, and an A/T-rich region downstream of the −10 region were kept constant, whereas sequences from −37 to −14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the −10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Eσ^s. The DNA sequence of these promoters differed significantly in the region from −25 to −16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

Promoters derived from banana bunchy top virus DNA-1 to −5 direct vascular-associated expression in transgenic banana (Musa spp.)

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants. Received: 17 May 1999 / Revision received: 3 November 1999 / Accepted: 4 November 1999     

Engineering of a Sagiyama alphavirus RNA-based transient expression vector

Sagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full-length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N-terminal 9 amino acids and C-terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N-terminally-fused capsid-protease domain. GFP was also expressed efficiently without the 5'-terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly-to-Leu substitution at the - 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 pg from 1 X 10(6) BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high-level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.     

茶碱激活型RNA分子开关的构建及在枯草芽胞杆菌中调节外源基因表达的性能

枯草芽胞杆菌Bacillussubtilis在工业生物技术以及合成生物学领域作为一种重要的微生物可广泛用作代谢工程、重组蛋白表达以及新型基因电路的底盘。在B. subtilis中构建基于非编码RNA的高精准调节元件,能够实现不依赖蛋白质因子的基因表达调控,丰富B.subtilis基因表达通用工具。通过基因工程手段,设计了基于茶碱适体域的核糖开关E和适体核酶AZ调节元件,并与不同的B.subtilis内源组成型启动子适配,构建出茶碱激活型基因表达控制元件。测定这两种调节元件与6种组成型启动子组合匹配下报告基因GFP的荧光强度,鉴定并分析各调控元件的工作性能。并进一步以红色荧光蛋白mCherry和普鲁兰酶两种不同的异源蛋白验证核糖开关或适体核酶与启动子的最优组合。结果表明,同一种RNA调节元件与不同启动子组合呈现不同水平的调控效率。在核糖开关与启动子的组合中,启动子PsigW和核糖开关E组合(sigWE)对GFP表达的诱导率最高,达到16.8。在适体核酶与启动子的组合中,AZ与启动子P43、PrpoB组合(P43AZ和rpoBAZ)的诱导率最高,分别达到了6.1和6.2。进一步验证结果显示,sigWE调控mCherry的诱导率最高(9.2),而P43E调控普鲁兰酶的诱导率最高(32.8),产酶水平达到了81U/mL。核糖开关和适体核酶对GFP、mCherry、普鲁兰酶均能实现调控,但是不同元件组合的调控性能有所差异,对不同基因的调控效果也不尽相同。     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.