抗砷载体的构建及在喜温硫杆菌中的接合转移

利用DNA体外重组技术,将质粒载体pUM3上的抗砷基因片段亚克隆到含有强启动子(tae启动子)并具有广泛寄主范围特性的IncQ族质粒pMMB24上,成功构建了含有强启动子的抗砷质粒pSDRA3,以及删除调节基因片段的组成型表达的抗砷质粒pSDRA4。通过接合转移的方式将其导入专性自养极端嗜酸性的喜温硫杆菌中,首次成功地建立了喜温硫杆菌的遗传转移系统,构建了冶金工程菌T.caldus(pSDRA3)和T．caldus(pSDRA4)。与野生菌相比,重组菌抗砷性能明显提高。     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对其细胞生长及抗生素合成的影响

肉桂地链霉菌(S.cinnamonensis)是莫能菌素(Monensin)的产生菌。大肠杆菌链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因(vhb)位于硫链丝菌素诱导启动子P_tipA之下,它在肉桂地链霉菌中的结构不稳定,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分和vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的VHb蛋白。摇瓶发酵实验证明,VHb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

酿酒酵母gpd1和hor2基因在大肠杆菌中的共表达

利用途径工程的方法,在大肠杆菌中构建一条新的产甘油的代谢途径。从酿酒酵母(Saccharomycescerevisiae)克隆3_磷酸甘油脱氢酶基因(gpd1)和3_磷酸甘油酯酶基因(hor2 ) ,并将两个基因串连到启动子trc的下游,构建由trc启动子控制的能高效表达的多顺反子重组质粒pSE_gpd1_hor2 ,将重组质粒导入大肠杆菌BL2 1菌株中,构建得到的重组菌株GxB_gh能将葡萄糖转化为甘油。结果表明重组菌株GxB_gh以葡萄糖为底物进行发酵,甘油产量为4 6 6 7g L ,葡萄糖的转化率为4 2 87%。这为利用工程菌绿色生产甘油进行了前期的探索,也为进一步构建能生产1,3_丙二醇的工程菌打下了良好的基础。     

多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrum sp. Mp4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS5载体上,构建重组质粒pTPD,在辅助质粒pRK2013 的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putida KT2440中,获得的工程菌Pseudomonas putida KT2440DOP可以降解多种有机磷农药及芳香烃化合物;KT2440DOP的有机磷水解酶活较出发菌株MP4提高了一倍左右,且遗传性状稳定。     

大肠杆菌-链霉菌高效接合载体的构建及其应用

以链霉菌质粒SCP2 的衍生质粒pHJL400为基础 ,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒pGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点 ,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化EscherichiacoliET12567(pUZ8002 )后 ,与天蓝链霉菌 (StreptomycescoelicolorA3(2 ) )、除虫链霉菌 (Streptomycesavermitilis)、变铅青链霉菌 (StreptomyceslividansTK54 )、毒三素链霉菌 (StreptomycestoxytriciniNRRL15443)、委内瑞拉链霉菌 (Streptomyces.venezuelaeISP5230 )和红色糖多孢菌 (Saccharopolyporaerythraea)进行接合 ,发现本文构建的pGH112与pKC1139相比 ,接合转移效率较高 ,稳定性好 ,而且宿主范围较广。把组成型启动子ermE 与绿色荧光蛋白基因 (gfp)克隆到本文构建的pGH112 ,通过接合转移到链霉菌中 ,gfp获得表达,证明其可以用作基因接合转移的有效工具载体,这为研究链霉菌的基因功能创造了有利条件上。     

绿色荧光蛋白基因标记野生型生防枯草芽孢杆菌的研究

根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR 序列,分别设计两对特异引物primers PxyF/R和primers gfpF/R,扩增获得了完整的启动子PxylR和-gfp基因序列。进一步以上述产物混合物为模板,以primer PxyF/primer gfpR做引物进行重迭PCR,获得了PxylR-gfp重组翻译融合表达盒。经SphⅠ和KpnⅠ完全酶切后,将PxylR-gfp表达盒分别插入大肠杆菌_苏云金芽孢杆菌穿梭载体pHT315和大肠杆菌枯草芽孢杆菌穿梭载体pRP22。相应的重组表达质粒pGFP315和 pGFP22转化枯草芽孢杆菌感受态细胞。前者在标准菌株168中得到良好发光表型,后者则在标准菌株168和野生目标菌株B916中均得到良好的发光表型。室内平板抑菌实验结果显示B916生防效果与出发菌株没有明显差异,遗传稳定性研究表明连续稀释培养约175代后,工程菌株稳定性为94%,质粒丢失频率低于3.5×10^-4/代。     

携带SHV类超广谱β-内酰胺酶的吉戈菲肠杆菌北京株的研究

用接合转移的遗传学方法证实了临床分离菌株吉戈菲肠杆菌(Enterobactergergoviae)3773含有一约60kb的可转移质粒,又用测耐药表型、酶水解率及基因片段杂交等方法证实了该质粒上有一编码产生超广谱β-内酰胺酶(Esbla)的基因.其大肠杆菌接合子除了头霉甲氧塞吩(cefoxitin)和亚胺硫霉素(imipenem)外,几乎对所有测定的β-内酰胺类药物都表现耐药,β-内酰胺酸抑制剂——棒酸(clavulanate)可抑制此Esbla的活性.携带此Esbla基因的质粒的一片段可与SHV-1的一结构基因片段杂交,说明此酶是SHV类的Esbla.     

通过接合作用质粒从大肠杆菌到棒状杆菌的转移

构建了可接合转移的穿梭质粒pXZ911、pBZ51和pBZ52。这些质粒中含有具转移功能的Mob片段和在棒状杆菌中复制的复制区。以大肠杆菌S17—1为供体菌,通过接合转移作用,可将这些质粒转移到谷氨酸棒杆菌ATCCl3032、谷氨酸棒杆菌ATCC21543、北京棒杆菌B3、北京棒杆菌1．299、裂氏棒杆菌B43、黄色短杆菌ATCC 14067等棒状杆菌苗株,接合转移频率分别为：9X10^-5,1X10^-4,8．5x10,2．3X10^-4×10^-5,2．9X10^-5。本文还探索了大肠杆菌和几种革兰氏阳性棒状杆菌问基因转移的方法、转移频率、影响转移频率的因素、宿主范围等问题。     

用基于“Neo/E”的技术构建重组质粒

根据重组工程原理,建立了一种用于构建重组质粒的 “neo/E”（抗生素/单酶切位点）选择与反选择新方法。首先采用 PCR方法扩增出线性打靶分子：然后进行两步体内同源重组,（1）neo/E基因敲入,重组子呈现neo抗性表型;（2）目的基因替换neo/E基因。用限制酶E消化时,发生第二步重组的DNA分子不能被消化,能够转化大肠杆菌受体菌DH5α。应用该方法构建了重组质粒pGL3-Basic PC1900T。PCR及测序鉴定证明：外源片段重组率为20%,所建立的重组工程选择与反选择新技术为质粒构建提供了新的解决方案。     

耐盐及苯乙酸、甲基对硫磷降解基因工程菌的构建

H1（Halomonas sp.）是一株耐高盐浓度（18% NaCl, W/V）和降解苯乙酸的菌株,pDT3质粒为pUC19插入甲基对硫磷水解酶基因（mpd基因）构建而成。采用ＨindⅢ酶切,获得含有完整mpd基因片段,克隆到广宿主质粒pKT230和pBBR1MCS2上,构建成质粒pKTMP和pBBRMP。通过三亲杂交,在辅助质粒pRK2013的帮助下,将质粒pKTMP和pBBRMP转移到Ｈ１中,得到的工程菌HpKTMP和HpBBRMP具有耐盐、降解苯乙酸和水解甲基对硫磷的功能,其中HpBBRMP水解酶活性与亲本菌株甲基对硫磷降解菌（Pseudomonas putida）DLLE4相当,而HpKTMP水解酶活性要提高1倍左右。经过传代试验,证明了工程菌的稳定性。     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.     

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.     

大肠杆菌磷酸果糖激酶基因在极端嗜酸性氧化硫硫杆菌中的表达

构建了含大肠杆菌磷酸果糖激酶(EC 2.7.1.11)基因pfkA的重组质粒pSDK1,利用大肠杆菌pfk缺陷株筛选含目的基因的重组质粒,通过接合转移的方式将其导入氧化硫硫杆菌TtZ2中,接合转移频率达2.6×10-6。重组质粒在TtZ2中有较好的稳定性,在无选择压力条件下传代50次基本保持稳定(重组质粒保留68%以上)。酶活性测定、SDSPAGE及RTPCR结果表明,pfkA基因在氧化硫硫杆菌中得到表达,但其表达水平低于大肠杆菌。葡萄糖可促进含pSDK1的氧化硫硫杆菌TtZ2的生长,而对照菌株的生长则未受明显影响,说明重组菌可部分利用葡萄糖作为碳源生长。     

泛耐药基因NDM-1在大肠杆菌中的克隆表达及耐药性检测

目的:构建bla(NDM-1)基因重组质粒,表达新德里金属β内酰胺酶1(NDM-1),并检测携带bla(NDM-1)基因重组质粒的大肠杆菌的耐药状况。方法:PCR扩增编码NDM-1的基因bla(NDM-1),构建表达载体pGEX4T-1-NDM-1,并转化至大肠杆菌,转化子经PCR后测序,以确认构建和转化成功;用Western印迹验证重组蛋白的表达;用药敏纸片法检测含重组质粒pGEX4T-1-NDM-1的大肠杆菌的耐药谱;用E-test法测定其最低抑菌浓度(MIC)。结果:PCR及测序结果显示载体构建和转化成功;含重组质粒pGEX4T-1-NDM-1的大肠杆菌在37℃时,经1 mmol/LIPTG诱导5 h后,SDS-PAGE可见目的条带;除对替加环素和粘菌素敏感外,该重组子对多种碳青霉烯类抗生素耐药,E-test检测其对亚胺培南的MIC为64μg/mL。结论:构建了含泛耐药基因bla(NDM-1)的重组质粒,转入大肠杆菌后表达了融合蛋白,并对多种碳青霉烯类抗生素耐药。为进一步研究bla(NDM-1)基因和蛋白的功能奠定了基础。     

大肠杆菌-链霉菌高效接合载体的构建及其应用

以链霉菌质粒SCP2^*的衍生质粒pHJL400为基础,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒DGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化Escherichia coli ET12567(pUZ8002)后,与天蓝链霉菌(Streptomyces coelicolor A3(2))、除虫链霉菌(Streptomyces avermitilis)、变铅青链霉菌(Streptomyces lividans TK54)、毒三素链霉菌(Streptomyces toxytricini NRRL15443)、委内瑞拉链霉菌(Streptomyces．vertezuelae ISP5230)和红色糖多孢菌(Saccharopolypora erythraea)进行接合,发现本构建的pGH112与pKC1139相比,接合转移效率较高,稳定性好,而且宿主范围较广。把组成型启动子ermE^*与绿色荧光蛋白基因(gfp)克隆到本构建的pGH112,通过接合转移到链霉菌中,gfp获得表达,证明其可以用作基因接合转移的有效工具载体,这为研究链霉菌的基因功能创造了有利条件。     

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus

Arsenical resistance is important to bioleaching microorganisms because these organisms release arsenic from minerals such as arsenopyrite during bioleaching. The acidophile Acidithiobacillus caldus KU was found to be resistant to the arsenical ions arsenate, arsenite, and antimony via an inducible, chromosomally encoded resistance mechanism. Because no apparent alteration of the toxic ions was observed, Acidithiobacillus (At.) caldus was tested to determine if it was resistant as a result of decreased accumulation of toxic ions. Reduced accumulation of arsenate and arsenite by induced At. caldus cells supported this hypothesis. It was also found that, with the addition of an energy source, induced At. caldus could transport arsenate and arsenite out of the cell against a concentration gradient. The lack of efflux in the absence of an added energy source and in the presence of inhibitors suggested that efflux was energy dependent. Induced At. caldus also expressed arsenate reductase activity, indicating that At. caldus has an arsenical resistance mechanism that is analogous to previously described systems from other Bacteria. Southern hybridization analysis showed that At. caldus and other gram-negative acidophiles carry an Escherichia coli arsB homologue on the chromosome.     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.     

Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.