A cryptic plasmid from Shigella sonnei

pNZ500 is a 1.5 kb cryptic plasmid from a Shigella sonnei isolate. It was introduced into Escherichia coli by cotransformation, where it is maintained at about 30 copies per chromosome equivalent. Hybridization studies show that pNZ500 exhibits a high level of sequence similarity to other 1.5 kb plasmids found in different S. sonnei isolates but shares no homology with larger S. sonnei plasmids. pNZ500 shares a small degree of sequence homology with pBR322 and with pAC184. The homology with pBR322 is restricted to sequences close to the ori-bom region of this plasmid. Nevertheless, pNZ500 maintenance in E. coli is not dependent on DNA polymerase I activity, and does depend on continuing protein synthesis. pNZ500 encodes two polypeptide gene products whose monomer molecular weights are 24500 and 18000. The examination of host cells for the expression of possible plasmid phenotypes revealed no differences between cells bearing pNZ500 and plasmidless cells.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从弗氏柠檬酸细菌（Citrobacter freundii)中扩增得到含有酪氨酸酚解酶基因的DNA片段,定向连续到质粒pUC118上,得到重组质粒pTPL,将此重组质粒转化到受体菌E.colXL-1-Blue MRF′中,通过蓝白斑鉴定挑出阳性菌株。从此阳性菌株中提取质粒pTPL并将此质粒转入到E.coliJM109中,用E.coliJM109（pTPL)制备高活性的酪氨酸酚解酶。对质粒稳定性的研究表明,E.coliJM109（pTPL)在无选择压力下37℃连续培养50代以上,质粒丢失率仅有15％,说明质粒基本稳定。     

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。     

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.     

苏云金芽胞杆菌杀蚊基因在鱼腥藻中克隆和表达的初步研究

将苏云金芽胞杆菌以色列亚种的杀蚊晶体蛋白基因cry11A亚克隆到大肠杆菌－蓝藻的穿梭质粒载体pRL25C,然后用三亲本杂交的方法将重组质粒转移到一种具有固氮能力且可被蚊幼虫吞食的鱼腥藻（Anabaena）PCC7120中。Southernblot及Westernblot分析表明cry11A基因在鱼腥藻PCC7120中得以克隆和表达,但生物测定未能检测到转基因鱼腥藻对库蚊（Culex）的毒性,可能是因为带有苏云金芽胞杆菌自身启动子的Cry11A基因在鱼腥藻PCC7120中表达量不够高的缘故。     

HCV core 1b亚型和HA共表达重组腺病毒载体的构建与鉴定

目的：构建HCV core 1b亚型和HA共表达的腺病毒载体并予以鉴定。方法：合成HCV核心蛋白1b亚型的核苷酸,连接入pcmv5-HA质粒中。用XhoⅠ单酶切,后用Klenow酶补平,再用HindⅢ单酶切下HA-HCV core 1b片段,连入pShuttle-cmv穿梭质粒中,构建穿梭质粒pShuttle-CMV-HA core 1b.将pShuttle-CMV-HA core 1b转化至含有AdEasy-1的BJ5183感受态细菌中进行同源重组。PacⅠ酶切线性化重组质粒pAd-HA-HCV core 1b并转染AD293细胞进行病毒包装和扩增。用RT-PCR检测HA-HCVcore 1b的mRNA的表达水平,并用Western blot检测融合表达蛋白HA-HCV core 1b的表达水平。结果：穿梭质粒pShuttle-CMV-HA-HCV core 1b经PCR和测序证实构建成功。重组腺病毒载体经AD293细胞包装后,可观察到CPE现象。用获得的重组腺病毒载体感染AD293细胞,经RT-PCR检测,在mRNA水平上有表达;经Western blot检测,HCV core 1b亚型腺病毒载体（Ad-HA-HCV core 1b）感染组与未感染腺病毒载体组及空白对照组相比,只有Ad-HA-HCV core 1b感染组有融合蛋白HA-HCVcore 1b的表达。结论：通过分子克隆体外重组技术,成功构建了HCV core 1b亚型和HA共表达的重组腺病毒载体Ad-HA-HCVcore 1b。为进一步研究丙型肝炎病毒1b亚型引起丙肝感染中胰岛素抵抗的作用机制提供了方法。     

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.     

Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.     

耐辐射球菌pprI在大肠杆菌中表达增强细胞抗氧化能力的研究

将耐辐射球菌(Deinococcus radiodurans)与DNA修复有关的开关基因—pprI通过穿梭质粒pRADZ3导入大肠杆菌TG1中,使其在正常培养条件下(不需诱导剂)表达PprI蛋白,并通过Western blot证实该基因在TG1中可稳定表达。与转化了空白质粒pRADZ3 TG1对照,观察了改造后的两种大肠杆菌在有H2O2氧化压力下的存活率和大肠杆菌中两种过氧化氢酶（KatE, KatG）的活性表达差异。结果表明,无论在指数生长期还是稳定生长期,能表达PprI蛋白的大肠杆菌比对照的存活率要高出10％左右;非变性电泳结果表明,耐辐射球菌pprI 在大肠杆菌中的表达使得KatE活性在指数生长期与稳定生长期分别增加1.5～2倍和2.5～3倍。证明耐辐射球菌pprI 在大肠杆菌中的表达能够增强细胞抗氧化能力。     

在DH5α中拼接构建ADAM10全长真核表达载体的基因突变

目的:构建ADAMI0真核表达载体,为进一步研究其生物学功能打基础.方法:将人ADAM10的上下两部分基因片段(分别为全长基因的1 ～910bp和911 ～2 247bp片段),依次与真核表达载体pcDNA3.1相连,以大肠杆菌DH5α或BL21(DB)作为感受态宿主菌用于转化连接产物,拼接成全长的阳性克隆通过PCR、酶切和测序鉴定.结果:ADAM10下段基因与已正确连入上段的pcDNA3.1重组质粒拼接时,若用DH5α为感受态菌,则下半段出现碱基插入增加512bp,测序结果显示为ADAM10基因第1 531 bp～2 042 bp间的序列有紧邻的双份;若用BL21(DE3)为感受态,则无突变.结论:将ADAM10基因与pcDNA3.1真核表达载体依次拼接构建重组质粒时,以DH5α为宿主菌可出现基因序列增加的罕见突变,而以BL21(DE3)为宿主则无突变,由此成功构建ADAM10全长基因与pcDNA3.1的重组质粒.     

RecA-independence of the process of amplification caused by the RS-1 sequence of Vibrio cholerae

The processes of amplification and deamplification of a plasmid DNA segment flanked by direct repeats of RSI-sequence of Vibrio cholerae and carrying the structural genes of cholera toxin inside the recombinant plasmid in E. coli cells have been studied. These processes determined by RSI-sequences are shown to take place independent of the RecA-system of E. coli cells.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes.

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

大肠杆菌酸性磷酸酶基因克隆表达及应用

将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.