pGreen-S: A clone vector bearing absence of enhanced green fluorescent protein for screening recombinants

The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5α produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.     

Key Amino Acid Residues Responsible for the Color of Green and Yellow Fluorescent Proteins from the Coral Polyp Zoanthus sp.

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore.     

Escherichia coli expressing a green fluorescent protein (GFP) in Anopheles stephensi: a preliminary model for paratransgenesis

Issues, like emerging insecticide resistance in Anopheles mosquitoes, have led to a breakdown in many vector control programs. In this study, a recombinant Escherichia coli with plasmid expressing a green fluorescent protein (E.coli-GFP) was used as a paratransgenesis model to determine: the possibility of E. coli-GFP trans-stadial transmission. The effect of the water microflora, of bacteria-impregnated sugar solutions, and of blood-feeding on E. coli-GFP colonization and localization within An. stephensi tissues, were studied. The results demonstrated the persistence of E. coli-GFP during molting and metamorphosis events and its trans-stadial transmission. Also the efficacy of bacteria-impregnated sugar solutions for transferring the bacteria to the adult mosquito’s midgut was shown. A blood meal dramatically increased the number of bacteria within 24–48 h post feeding. In addition to fluorescence microscope evaluation, GFP gene PCR amplification showed the presence of the bacteria in the midgut of larvae, pupae, and adults up to 13 days after eclosion. Massive colonization of bacteria was observed in the larvae and in the adult mosquito’s malpighian tubules which may play a role in retaining bacteria in adult mosquitos. The results of this study showed that E. coli could be used as a laboratory model in paratransgenesis studies for the evaluation of various effector molecules as anti-parasite agents for malaria and filariasis.     

Construction of a New Bacterial Cloning Vector Using a Mutant Green Fluorescent Protein as an Indicator

A new bacterial cloning vector, pGreenLD, derived from the triple substitution mutated Aequorea v/ctor/a green fluorescent protein(GFP-S65A, V68L, S72A), when expressed in E. coli produced colonies which showed yellow-green colour under daylight and strong green fluorescence under long-wave ultraviolet light. It can be a useful vector for selecting foreign DNA fragment which was inserted into multiple cloning site based on the loss of the yellow-green color/green fluorescence of E. coli cells attributable to the insertional inactivation of GFP production.     

利用突变的绿色荧光蛋白基因为标记构建新型克隆载体

将三位点替换突变的绿色荧光蛋白（ＧＦＰ＿Ｓ６５Ｔ、Ｖ６８Ｌ、Ｓ７２Ａ）基因插入到ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＸｂａⅠ和ＳａｃⅠ之间,构建为新型的克隆载体ｐＧｒｅｅｎＬＤ。此质粒载体在Ｅ．ｃｏｌｉ中表达后使菌落在日光下呈现黄绿色,而在长波紫外光照射下呈现亮绿荧光,当外源ＤＮＡ片段插入该载体的多克隆位点使ｇｆｐ基因表达受阻时,转化的Ｅ．ｃｏｌｉ菌落为白色。因此可以用Ｅ．ｃｏｌｉ菌落黄绿色／绿色荧光的消失作为检测指标来筛选含有重组质粒的克隆。     

<Emphasis Type="BoldItalic">In situ</Emphasis> real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe <Emphasis Type="BoldItalic">Deinococcus radiodurans</Emphasis>

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.     

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.     

Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Reconstruction of the Carotenoid Biosynthetic Pathway of Cronobacter sakazakii BAA894 in Escherichia coli

Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894.     

A genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>, expressing the fusion protein of green fluorescent protein and carboxylesterase B1, can be easily detected in the environment following degradation of pesticide residues

Genetically engineered Escherichia coli, expressing the fusion protein of enhanced green fluorescent protein (EGFP) and carboxylesterase B1 (CarE B1), was successfully constructed by cloning the genes into the pET-28b vector and then transforming E. coli BL21 (DE3). Expression of the fusion protein was induced in E. coli BL21 (DE3) which could then degrade environmental pesticides and could be easily detected using fluorescence spectrophotometry or by the naked eye in daylight.     

Optical properties of the monomeric red fluorescent protein mRFP1

For the expression in E. coli, the PCR-amplified fragment encoding mRFP1 from vector pMT-mRFP1 (Fungal Genetic Stock Center) was placed in the pQE-60 vector. Chemically competent E. coli ER1821 were transformed and grown overnight at 37°C. The protein was purified by Ni-NTA chromatography and dialyzed against 67mM Na₂HPO₄, 67mM KH₂PO₄, pH 7.5. There are two peaks (at 503 and 584 nm) in the mRFP1 absorption spectrum. The green component (503 nm) may correspond to a green fraction of the protein (a fraction that never matures beyond the green intermediate or a fraction which is trapped as a dead-end product such as the nonproductive trans conformation for the F65-Q66 peptide bond). The mRFP1’s extinction coefficient is equal to 42 mM^?1 cm^?1 at 584 nm; the emission maximum is at 607 nm; the excitation maximum is at 584–586 nm; the Stokes shift is equal to 23 nm; the fluorescence lifetime is about 1.8 ns; the quantum yield is 0.27; pKa is 4.0. Analysis of the mRFP1 absorption spectrum by high-order derivative spectroscopy shows that electron transition systems of both the fully matured form (absorption maximum at 584 nm) and the green fraction of the protein (absorption maximum at 503 nm) are practically identical.     

Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5α and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 μM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 μM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Mistranslation Reduces Mutation Load in Evolving Proteins through Negative Epistasis with DNA Mutations

Translational errors during protein synthesis cause phenotypic mutations that are several orders of magnitude more frequent than DNA mutations. Such phenotypic mutations may affect adaptive evolution through their interactions with DNA mutations. To study how mistranslation may affect the adaptive evolution of evolving proteins, we evolved populations of green fluorescent protein (GFP) in either high-mistranslation or low-mistranslation Escherichia coli hosts. In both hosts, we first evolved GFP under purifying selection for the ancestral phenotype green fluorescence, and then under directional selection toward the new phenotype yellow fluorescence. High-mistranslation populations evolved modestly higher yellow fluorescence during each generation of evolution than low-mistranslation populations. We demonstrate by high-throughput sequencing that elevated mistranslation reduced the accumulation of deleterious DNA mutations under both purifying and directional selection. It did so by amplifying the fitness effects of deleterious DNA mutations through negative epistasis with phenotypic mutations. In contrast, mistranslation did not affect the incidence of beneficial mutations. Our findings show that phenotypic mutations interact epistatically with DNA mutations. By reducing a population’s mutation load, mistranslation can affect an important determinant of evolvability.     

龋齿致病菌变形链球菌蛋白 Smu.260 的 制备、结晶及初级晶体学分析

变形链球菌 (Streptococcus mutans) 是最主要的龋齿致病菌,其基因 Smu.260 编码一个约 23 ku (200 个氨基酸 ) 的蛋白质. Smu.260的 DNA 片段被克隆到表达载体 pET28a 后在大肠杆菌 BL21(DE3) 菌株中表达得到很好的产量. 产物 Smu.260 蛋白通过 Ni²⁺亲和柱和分子筛两步法纯化,并发现纯化后的蛋白以两种形式存在,二聚体 (约46 ku) 和四聚体,前者呈亮黄色,后者无色. 采用悬滴气象扩散法得到了二聚体形式的晶体. 晶体的 X 射线衍射分辨率达到 2.3埃,晶体属正交空间群 P2₁2₁2₁,晶格参数为a=89.88埃, b=90.91埃, c=105.17埃. 晶胞不对称单元内估计含有一个二聚体,溶剂含量为 53％ .     

PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10⁶ transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.     

DNA modification and functional delivery into human cells using Escherichia coli DH10B

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ~200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd^– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.     

Fate of Escherichia coli during Ensiling of Wheat and Corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 × 10⁸ CFU/g into direct-cut wheat (348 g of dry matter kg⁻¹), wilted wheat (450 g of dry matter kg⁻¹), and corn (375 g of dry matter kg⁻¹). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens

The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.