Development of a versatile shuttle vector for gene expression in Geobacillus spp

An improved, versatile shuttle vector has been created for the metabolic engineering of Geobacillus spp. As kanamycin is the most thermo-tolerant of commonly used antibiotics, the gene encoding a thermostable kanamycin nucleotidyltransferase, together with the origin of replication from the G. stearothermophilus plasmid pBST1 were cloned into the Escherichia coli cloning vector pUC18. The resulting vector, named pUCG18, replicated in both organisms and could be transformed with an efficiency of 1 x 10(4) transformants per microg of DNA in G. thermoglucosidasius and was stable up to 68 degrees C with antibiotic selection. It was used to demonstrate expression of the pyruvate decarboxylase (pdc) gene from Zymomonas palmae in G. thermoglucosidasius at 45 degrees C. Sequence analysis of the pBST1 derived origin of replication revealed homology with a family of theta replicons that have previously only been found in strains of Bacillus megaterium.     

Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp.     

Construction of an inducible expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis

An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.     

Construction of two vectors for gene expression in Trichoderma reesei

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

Construction of a high-efficiency shuttle vector for Histophilus somni

The genetic manipulation of Histophilus somni is limited due to its high-fidelity restriction-modification system. The broad host-range shuttle plasmid pLS88 is capable of transforming some strains of H. somni, but is an inefficient vector. We have constructed an improved version of pLS88, pNS3K, that transforms H. somni strain 2336 100-fold more efficiently than its predecessor. The transformation efficiency was further increased when pNS3K was isolated from H. somni and retransformed into the same strain. As proof of principle, the lipooligosaccharide biosynthesis gene lob-2A was cloned into pNS3K and expressed in H. somni strain 129Pt, which lacks this gene. Thus, pNS3K is a useful shuttle vector for H. somni and a potential vector for genetic manipulation of this bacterium.     

Expression of the S. aureus hysA gene in S. carnosus from a modified E. coli-staphylococcal shuttle vector

We have modified an E. coli-staphylococcal shuttle vector for use in the general cloning and expression of genes from pathogenic staphylococci in Staphylococcus carnosus. As S. carnosus is non-pathogenic, this expression system will facilitate the study of the roles of individual gene products in the disease process. To evaluate the use of this expression system, a DNA fragment containing the Staphylococcus aureus hyaluronate lyase (hysA) gene was cloned into the modified vector, pNW21, and introduced into S. carnosus. Hyaluronate lyase was both produced and secreted by S. carnosus. In addition, the secreted HysA protein was enzymatically active, as determined using a zymographic assay.     

pPSY: a vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividansamyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli.     

Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was > 34 kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5 kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5 hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.     

Low copy number plasmid vectors for gene cloning and for monitoring gene expression

Abstract Derivatives of an IncW incompatibility plasmid with a low copy number are described which can be used for gene cloning or for analysing gene expression in conditions similar to those found in the host chromosome. Gene expression can be monitored after construction of operon or protein fusions with the lacZYA operon and measurement in Escherichia coli of the β-galactosidase activity.     

Detection of copy number variations and their effects in Chinese bulls

Background

Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.

Results

We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.

Conclusions

The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-480) contains supplementary material, which is available to authorized users.     

Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis

The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The lacIq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.     

Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum

Bacillus subtilis sacB gene with its 463 bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilissacB with the PlacM fused at the 5′-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.     

极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用

摘要：【目的】利用有自主知识产权的嗜盐古菌θ型复制质粒和启动子,构建在极端嗜盐古菌模式菌株西班牙盐盒菌（Haloarcula hispanica）中方便使用、功能完善的基因表达载体。【方法】以pSCM201的最小复制子为基础,通过引入莫维诺林抗性基因,大肠杆菌质粒复制子以及氨苄抗性基因,构建了一个新的嗜盐古菌-大肠杆菌穿梭载体。利用定点突变和末端补平法依次将其中多余的酶切位点去除后,再添加hsp5启动子核心序列、人工合成的多克隆位点以及蛋白纯化标签His?Tag等重要元件成功构建了嗜盐古菌表达载体pSCM307。将报告基因bgaH插入到该载体的多克隆位点中并转化H. hispanica AS2049,通过X-gal平板筛选和β-半乳糖苷酶酶活实验检测pSCM307的表达能力。【结果】pSCM307具有独立的自主复制能力,其多克隆位点方便实用,报告基因bgaH在hsp5启动子控制下实现了高效表达。【结论】成功构建了嗜盐古菌领域中第一个方便使用的基因表达载体。     

Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi

The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312 bp, a low G + C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the β-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB.     

Genomic DNA macroarrays as a tool for analysis of gene expression in Leishmania

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.