Construction of a Shuttle Vector for Use between Pasteurella multocida and Escherichia coli

The isolation of a small plasmid from Pasteurella multocida has enabled to construction of a shuttle vector for use between P. multocida and Escherichia coli. The vector pBAC64 contains the origin of replication from P. multocida, an antibiotic resistance gene which functions in P. multocida, and the E. coli vector pUC18. The presence of the pUC18 multiple cloning site together with the lacZ′ gene provides a screening method and allows cloning and manipulation in E. coli as well as cloning in P. multocida.     

大肠杆菌-枯草芽孢杆菌穿梭质粒的构建及碱性蛋白酶的表达

构建了一个可以在大肠杆菌和枯草芽孢杆菌中复制,并可以在枯草芽孢杆菌中表达外源蛋白的穿梭表达载体pBE2R.该穿梭表达载体是以pBE2为基本骨架,引入来自pWB980质粒的P43强启动子,并在其下游引入嗜碱芽孢杆菌碱性蛋白酶信号肽和前肽构建而成.试验结果表明,该穿梭表达载体pBE2R可以在大肠杆DH5α和枯草芽孢杆菌WB600中稳定存在,并可以使外源蛋白在枯草芽孢杆菌进行分泌表达,同时也为碱性蛋白酶定向改造提供了高通量筛选平台.     

Stable Escherichia coli-Clostridium acetobutylicum Shuttle Vector for Secretion of Murine Tumor Necrosis Factor Alpha

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-α) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-β1,4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-α cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-α during growth. Significant levels of biologically active mTNF-α were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.     

Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.     

大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达

目的:构建大肠杆菌-长双歧杆菌穿梭表达载体,并通过此载体使人内皮抑素基因在大肠杆菌和长双歧杆菌中得到表达。方法:以质粒pDG7、pBCSK( )、pET-9C为基础,构建大肠杆菌-长双歧杆菌穿梭表达载体pET-1128,并将人内皮抑素基因插入到新构建的表达载体中,分别转化大肠杆菌BL21(DE3)和长双歧杆菌NQ-1501。诱导表达,表达产物经SDS-PAGE和WesternBlot鉴定。结果:成功构建了大肠杆菌-双歧杆菌穿梭载体,人内皮抑素基因在大肠杆菌和长双歧杆菌中均可表达。结论:构建的穿梭载体为今后用双歧杆菌作为生理菌载体进行肿瘤的基因治疗奠定了基础。     

Construction of Novel Shuttle Vectors for Use between Moderately Halophilic Bacteria andEscherichia coli

Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid fromChromohalobacter marismortui,combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites,lacZfragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles. These vectors can be efficiently transferred by RP4-mediated conjugation fromEscherichia colito the moderate halophilesChromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola,andVolcaniella eurihalina.     

Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d-Lactate

Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stable Escherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer Leuconostoc citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using plasmid pMBLT02, which overexpresses d-lactate dehydrogenase, exhibited enhanced production of optically pure d-lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis.     

Construction of a New Shuttle Vector for Zymomonas mobilis

The culture conditions for Rhodococcus sp. N-774 cells showing high nitrile hydratase activity and the reaction conditions for acrylamide production by the resting cells were optimized. Thiamine was essential for the growth of the strain. Yeast extract and Fe^{2 +} or Fe^{3 +} remarkably promoted the formation of nitrile hydratase of the cells. The reaction proceeded optimally at temperatures below 30°C. Incubation for 1 hr at above 40°C resulted in inactivation of the enzyme. Through reaction at a temperature as low as 0°C, the inhibition and inactivation of the enzyme activity by the substrate, acrylonitrile, and the product, acrylamide, were remarkably reduced, and higher accumulation of acrylamide could be attained. Under the optimal conditions, a more than 20% (w/v) acrylamide solution was obtained with a conversion yield of nearly 100%. Thus, the aqueous acrylamide solution obtained showed a high enough quality for use for the commercial preparation of polyacrylamide.     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.     

Movement of Shuttle Plasmids from Escherichia coli into Yeasts Other Than Saccharomyces cerevisiae Using Trans-kingdom Conjugation

Transfer of bacteria/yeast shuttle plasmids from Escherichia coli into the yeast species Kluyveromyces lactis, Pichia angusta (Hansenula polymorpha), and Pachysolen tannophilus has been accomplished, presumably through inter-kingdom conjugal transfer. Plasmid pEK2 was transferred into a K. lactis mutant to complement trp auxotrophy, while plasmid YEp13 was mobilized into and complemented P. angusta and P. tannophilus Leu^- auxotrophs. Plasmid DNA in the recipient strains was detected by transformation of E. coli with crude yeast cell extracts. Freely replicating plasmids without detectable alterations as well as plasmids with rearrangements were recovered from yeast transconjugants.     

Characterization of the Stable Maintenance of theShigella flexneriPlasmid pHS-2

pHS-2 is a 3-kb plasmid originally isolated fromShigella flexneriinfections associated with reactive arthritis in humans. This plasmid is stably maintained in many clinical isolates ofShigella flexneri.The nucleotide sequence of this plasmid displays two closely linked regions that may play a role in the maintenance of this plasmid. One region consists of a 250-bp locus showing a significant homology to the ColE1cersite. The results indicate that thecer-like site of pHS-2, like the ColE1cersite, acts as arecA-independent, site-specific recombination site involved in the resolution of multimers, requiring the presence of the host-encoded factors ArgR, PepA, XerC, and XerD. The second region consists of a 36-kDa open reading frame involved in generating resistance to the bactericidal effect of complement, which confers a selective advantage to cells containing this sequence. The results also indicate that pHS-2 can replicate in another species of Enterobacteriaceae (Escherichia coli) and is mobilized by the F plasmid.     

Construction and Characterization of a Replication-Competent Retroviral Shuttle Vector Plasmid

We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.     

Nucleotide Sequence Determination and Genetic Analysis of theBacteroidesPlasmid, pBI143

The nucleotide sequence and genetic organization of theBacteroidesplasmid pBI143 were determined. The plasmid was 2747 base pairs (bp) and had a G+C content of 41% (GenBank Accession No. U30316). There were two open reading frames greater than 50 codons and these were designatedmobAandrepA.A 56-bp inverted repeat divided pBI143 into modules withrepAandmobAin separate regions. There was a marked difference in the G+C content and codon usage for the two regions;repAhad 33% G+C andmobAwas 44% G+C. MobA had homology to otherBacteroidesmobilization proteins and RepA shared homology to a replication protein fromZymomonas mobilisplasmid pZM2. These two putative replication proteins formed a subgroup of the rolling-circle replication proteins belonging to the pSN2 family of gram-positive plasmids. Consistent with this finding, single-stranded pBI143 DNA was detected in plasmid containingBacteroides fragiliscultures. Availability of the pBI143 sequence allowed the elucidation of the complete nucleotide sequence for pFD288 an 8.9-kbBacteroidesshuttle vector (GenBank Accession No. U30830).     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

链霉菌和大肠杆菌穿梭质粒载体的构建

本文报道了链霉菌和大肠杆菌穿梭质粒载体pSE-3的构建;把具有双启动子的大肠杆菌的质粒pGEM-3与新霉素抗性基因启动子缺失的链霉菌的探针质粒pIJ486分别用BamHI和BglⅡ酶切,T4 DNA连接酶连接后转化到E.coli HB101(Amp(?),Neo(?)),所得重组质粒能强启动pIJ486质粒上的氨基糖苷磷酸转移酶基因(aph),并使新霉素抗性基因在大肠杆菌中得到强表达。此重组质粒被命名为pSE-3,当其转化到变青链霉菌TK54(Tsr(?),Neo(?))的原生质体前,新霉素抗性基因亦能得到强表达。酶切结果表明,构建的具有两个启动子的穿梭质粒载体pSE-3上有HindⅢ和EcoRI的单酶位点,拷贝数约为39。经再转化和传代50代等研究表明,穿梭质粒载体pSE-3在链霉菌和大肠杆菌中均是稳定的。为某些有应用价值的目的基因在大肠杆菌和链霉菌中的克隆与表达提供了一个有价值的穿梭质粒载体。     

Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum

A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.