An Escherichia coli system for assay of Flp site-specific recombination on substrate plasmids

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the Flp site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40. pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying Flp-recognition targets (FRT) are transformed into BL-FLP, and the consequences of Flp-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect Flp-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used Flp substrate plasmid, pNEOβGAL (O'Gorman et al. (1991) Science 251, 1351–1355).     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

Restriction map of the hepatitis B virus DNA cloned in Escherichia coli

A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.     

大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

Release of outer membrane fragments from normally growing Escherichia coli

A complex containing lipopolysaccharides, phospholipids and proteins is released into the culture medium by Escherichia coli during normal growth. It can be separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation. Electron microscopy revealed that this material is released as vesicles and membrane fragments. To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes. The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane. Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.     

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells

Summary UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Purification and characterization of the -xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli -xylose isomerase ( -xylose ketol-isomerase, EC 5.3.1.5) gene and the -xylulokinase (ATP: -xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The -xylose isomerase gene was separated from the -xylulokinase gene by the construction of a new deletion plasmid, pLX7. The -xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the -xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first -xylose isomerase gene that has been isolated and extensively purified from any organism. -Xylose isomerase, the enzyme product of the -xylose isomerase gene, is responsible for the conversion of -xylose to -xylulose, as well as -glucose to -fructose. It is widely believed that yeast cannot ferment -xylose to ethanol primarily because of the lack of -xylose isomerase in yeast. -Xylose isomerase (also known as -glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the -xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting -xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.     

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.     

在大肠杆菌中表达有活性的人STK11蛋白

STK11蛋白(serine/threonine kinase11)是近年来发现的具有多种重要功能的蛋白,可参与调控细胞周期、p53介导的细胞凋亡、ras诱导的细胞转化、细胞极化等多种生物学过程。利用大肠杆菌高效表达有活性的人STK11蛋白,可为其结构和功能的深入研究打下良好基础。利用本室克隆的人STK11 cDNA和原核表达载体pET-44a( )构建带有Nus融合标签的诱导型表达载体pET-Nus-STK11,在不同的大肠杆菌宿主中诱导表达。SDS-PAGE和Western blot检测表明,在BL21(DE3)宿主中表达的融合蛋白主要以包涵体形式存在,占菌体总蛋白的8.9%;在Rosetta-gami(DE3)pLysS宿主中主要表达为可溶性蛋白,占菌体总蛋白的16.7%。而经纯化和包涵体蛋白复性处理后,以Chariot介导重组融合蛋白进入人肝癌细胞SMMC-7721检测其对细胞生长和细胞周期的影响。与对照组相比,BL21(DE3)中表达的Nus-STK11蛋白几乎无抑制活性;而Rosetta-gami(DE3)pLysS中表达的Nus-STK11蛋白可以显著抑制SMMC-7721细胞的生长,抑制率达47.05%,并导致细胞周期的G0/G1期阻滞,证实表达的重组融合蛋白具有明显的生物学活性。上述结果为在大肠杆菌中成功表达有活性的重组STK11蛋白的首次报道。     

利用重组大肠杆菌合成几丁寡糖的研究

提取根瘤菌Mesorhizobium.loti基因组,克隆编码N-乙酰氨基葡萄糖转移酶nodC基因,插入质粒pUC19的lac启动子的下游,构建并筛选出能够合成几丁寡糖的重组大肠杆菌DCL-3。利用优化的MMYNG培养基,重组大肠杆菌DCL-3在10L发酵罐中培养26h后,培养液菌体浓度测定OD560=10.8,几丁寡糖得率达到526mg/L。收集重组细菌的细胞并煮沸破碎,利用活性炭的吸附和P4凝胶层析对几丁寡糖产物进行分离纯化。纯化产物的液质分析(LC-ESI-MS)结果表明主要寡糖产物为几丁四糖(m/z,831[M H] )和几丁五糖(m/z,1034[M H] )。     

利用重组大肠杆菌进行寡糖合成的研究进展

随着更多寡糖生物学活性的阐明,寡糖合成研究已成为糖生物学研究的热点之一,其中,以重组大肠杆菌作为酶盒或生物反应器,利用Leloir途径合成寡糖的方法,是近年来发展起来的一类重要的寡糖生物合成技术,并取得了较多的进展。将从细菌糖基转移酶的表达和鉴定、糖核苷酸的供给和寡糖的合成途径等几个方面,关注利用细菌功能尤其是利用重组大肠杆菌合成寡糖的研究进展,并分析各技术手段的优缺点及其应用前景。     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Strategies for the Genetic Manipulation of Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries. In this article the plethora of methods available for manipulating the genome of S. cerevisiae are reviewed. This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods. Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries.