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.     

长双歧杆菌NCC2705高效转化系统的建立及GFP表达

目的:建立长双歧杆菌NCC2705高效稳定的电转化系统,并以其为宿主表达绿色荧光蛋白(GFP),构建一个稳定的带报告基因的表达载体。方法:从双歧杆菌克隆载体pDG7中切取其在双歧杆菌中复制所必需的pMB1复制子插入质粒pUC19的多克隆位点区,并将gfp基因在双歧杆菌中进行表达;设计双歧杆菌电击转化体系,研究在不同电击电压条件下的电转效率。结果:首先构建了大肠杆菌-长双歧杆菌穿梭质粒pUB1和带有gfp基因的表达质粒pUB2,用电击法将之转化至双歧杆菌,当细菌生长到D600nm约为0.5时制备感受态细胞,在电容25μF、电阻200Ω、电压12.5 kV/cm的电击条件下进行完整质粒转化,可得到较高的转化率。结论:构建了以长双歧杆菌NCC2705为宿主的高效稳定的表达系统,为进一步研究益生菌的分子生物学和功能特性提供了基础。     

Lambda phagemids and their transducing properties

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

大肠埃希菌—双歧杆菌穿梭表达载体的构建及白介素-10基因的表达

目的构建能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因的载体,并用此载体在大肠埃希菌和双歧杆菌中表达人白介素-10基因（hIL-10）的蛋白产物;为hIL-10基因重组双歧杆菌治疗炎症性肠病做前期准备。方法以质粒pDG7为模板扩增pMB1片段,构建表达质粒pET28B1。用PCR法扩增hIL-10基因,将此目的基因以及pET28B1经酶切后用连接酶连接,形成重组质粒pET28B1-hIL10。pET28B1-IL10转染大肠埃希菌BL21和长双歧杆菌。最后用Western blot检测hIL-10基因在大肠埃希菌和长双歧杆菌中的表达情况。结果pET28B1-hIL10阳性克隆扩增后提取质粒并进行基因测序,结果显示插入片段为hIL-10,序列正确且无突变。hIL-10基因在大肠埃希菌、长双歧杆菌中的诱导表达产物通过Western blot检测验证为IL-10蛋白,显示该hIL-10表达载体在大肠埃希菌阳性克隆中经诱导可高量表达,在长双歧杆菌体中有少量表达。结论成功构建质粒pET28B1,该质粒能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因hIL-10。     

Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon

This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.     

Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system

The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors.     

Replication of plasmids with the p15A origin in Shewanella putrefaciens MR-1

The plasmid pACYC184 was introduced into Shewanella putrefaciens MR-1 by electroporation. In 100% of the transformants examined, the plasmid was maintained as a free replicon outside the chromosome. This was the case whether or not the plasmid contained a 224-bp DNA insert derived from an open-reading frame of MR-1 genomic DNA. Therefore, in contrast to a report in the literature, plasmids containing the p15A origin of replication can replicate freely in S. putrefaciens MR-1, and do not make convenient vectors for gene replacement in this bacterium. However, we found that plasmids with the pMB1 origin of replication (e.g. pBR322) cannot replicate in MR-1 and could therefore have potential as vectors for gene replacement.     

A set of plasmids constitutively producing different RNA levels in Escherichia coli.

New plasmids were developed for the in vivo expression of RNA in Escherichia coli. These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA. The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions. The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors. Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter.     

A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)

A set of plasmid vectors conferring chloramphenicol resistance (Cm(R)), 3064bp in size, or kanamycin resistance (Km(R)), 2972bp in size, were developed, having multiple cloning sites in lacZ' genes for alpha-complementation. pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 are temperature-sensitive (ts) in DNA replication (ts-Rep); pTH18cs5, pTH19cs5, pTH18ks5 and pTH19ks5 are ts in plasmid segregation (ts-Seg); and pTH18cr, pTH19cr, pTH18kr and pTH19kr are temperature resistant (tr) in both. They are based on the pSC101 replicon consisting merely of the replication origin and repA gene, compatible with ColE1/pMB1/p15-derived plasmids, and thus do not require polA function of host cells. The copy numbers of the ts-Rep, tr and ts-Seg plasmids were 14, 5 and 1 per chromosome at 30 degrees C, respectively. These plasmids are fairly stable when inherited at 30 degrees C, but not above 37 degrees C or 41.5 degrees C, depending on the repA mutations and host strains. They are isogenic apart from the ts mutations in the repA gene, and thus provide with useful tools for having appropriate controls in various experiments including bacterial gene-targeting, transposon mutagenesis, toxic gene expression, differential substitution on host functions, gene dosage analysis and so on.     

Incompatibility properties of Col E1 and pMB1 derivative plasmids: Random replication of multicopy replicons

The incompatibility properties of Col E1-like plasmids have been examined in Rec⁺ and RecA^? bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.     

Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7.

The Mycobacterium avium plasmid pLR7 is representative of a group of small plasmids that are common in isolates from AIDS patients with disseminated M. avium infections. Determination of the functions of these and other plasmids has been hampered by the lack of methods for genetic manipulation of M. avium. In this study, the region of pLR7 capable of replication was identified and sequenced. Fragments of pLR7 were cloned into a pUC18 derivative carrying a kanamycin resistance marker and introduced into a plasmid-free M. avium strain by electroporation. The origin of replication was located on a 1.8-kb PvuII-to-SmaI fragment. An open reading frame encoding a putative Rep protein was identified. Two other open reading frames were identified in this region. A shuttle vector, pMB351, was constructed with the pLR7 origin of replication, pUC18, and the kanamycin resistance gene from Tn5. This vector was successfully transformed into M. avium, Mycobacterium tuberculosis, and Mycobacterium bovis.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

苏云金芽胞杆菌拟步行甲亚种质粒复制子oril65的克隆

以苏云金芽胞杆菌拟步行甲亚种菌株（Bacillus thuringiensis subsp.tenebrionis)YBT-1765作为出发菌株,克隆了一个包含复制子的EcoRI酶切片段,大小约为11kb,称为oril65。这是国内外从此亚种中克隆到的第一个复制子,缩小到8kb左右后仍然能够复制。杂交结果显示,此复制子来源于菌株YBT-1765可以检测到的分子量最大的质粒,以此复制子构建的穿梭载体pBMB6071在不同受体菌中的稳定性差异很大,其中在以色列亚种无晶体突变株4Q7中,传40后代,稳定性100%,质粒pBMB6071与含ori1030和ori2062在库斯塔克亚种无晶体突变株BMB171中是相容的。     

Cloning and characterization of 4.5S and 5S RNA genes in tobacco chloroplasts

Tobacco chloroplast 4.5S and 5S RNAs were shown to hybridize with a 0.9 . 10(6) dalton EcoRI fragment of tobacco chloroplast DNA. Recombinant plasmids were constructed from fragments produced by partial digestion of the chloroplast DNA with EcoRI and the pMB9 plasmid as a vector. Five recombinants containing the 4.5S and 5S genes were selected by the colony hybridization technique. One of these plasmids contained also the 16S and 23S RNA genes and was mapped using several restriction endonucleases as well as DNA-RNA hybridization. The order of rRNA genes is 16S-23S-4.5S-5S and the four rRNA genes are coded for by the same DNA strand.