大肠杆菌Zn2+敏感突变株构建及其功能验证

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用,Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中,zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株,并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株,利用λ Red重组系统,通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上,利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因,获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E. coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件,同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠...     

大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

大肠杆菌BL21(DE3)lpxM突变株的构建

目的:lpxM基因失活可以产生极低内毒素活性的脂多糖。构建大肠杆菌BL21(DE3)的lpxM突变株,并考察该突变株的生长状态和表达重组蛋白的能力。方法:构建同源臂长500bp左右的打靶载体,借助Red同源重组系统,使E.coliBL21(DE3)的lpxM基因发生插入失活,再导入编码FLP位点特异性重组酶的质粒pCP20去除抗性基因。PCR鉴定发生插入突变的菌株,应用SDS-PAGE分析突变前后的脂多糖,考查对重组蛋白表达的影响。结果:PCR鉴定结果说明lpxM基因发生了插入突变。与出发株比较,突变株的脂多糖电泳图谱发生了明显变化,但其生长状态与表达重组蛋白的能力与出发株基本一致。结论:大肠杆菌BL21(DE3)的lpxM突变株可以用于重组蛋白的表达。     

大肠杆菌O157菌体抗原基因检测芯片的制备

选择编码O157菌体抗原特异合成酶的rfbE基因设计引物于口探针,并制备检测芯片,通过两次PCR扩增,制备荧光标记的靶序列,并与芯片进行杂交,检测O157菌株和非O157病原体。结果所有O157菌株均在芯片相应探针处出现阳性信号,非O157杂交结果均为阴性;芯片检测灵敏度比PCR检测高50倍。说明基因芯片可以快速、灵敏、特异地检测O157菌体抗原,为建立快速灵敏的检测细菌病原体特征和鉴别诊断的自动分析系统提供了新方法。     

应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后,生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型。【结论】在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法。     

大肠杆菌menA敲除对CoQ积累的影响研究北大核心CSCD

通过同源重组敲除大肠杆菌的men A基因,增加菌体Co Q合成量,用于构建Co Q高产菌株。以p KD4质粒为模板,PCR扩增kanr片段;在p KD46的辅助下,kanr片段转化大肠杆菌,利用抗生素筛选和PCR验证重组子;以紫外诱变菌为对照,发酵men A基因敲除菌株,分析Co Q种类和产量变化。成功获得men A基因敲除菌株,Co Q种类不变,产量增加约为38%。首次敲除men A基因,改良株Co Q产量得到提高,达到预期目标。     

大肠杆菌酪氨酸转运系统基因敲除对酪氨酸生产的影响

酪氨酸是三大芳香族氨基酸之一,广泛用于食品、医药和化工等领域。转运系统工程为代谢工程改造大肠杆菌选育酪氨酸生产菌株提供了一种重要的研究策略。大肠杆菌中酪氨酸胞内转运主要通过aroP和tyrP基因编码的通透酶进行调控。以酪氨酸生产菌株HGXP为出发菌株,利用CRISPR-Cas9技术成功构建了aroP和tyrP基因敲除菌,并通过发酵试验考察了调节转运系统对酪氨酸生产的影响。发酵结果表明,aroP和tyrP基因敲除菌酪氨酸产量分别达到3.74 g/L和3.45 g/L,较出发菌株酪氨酸产量分别提高了19%和10%。对诱导温度进行了优化,结果表明38℃为最佳诱导温度。在3 L发酵罐上进行了补料分批发酵,aroP和tyrP基因敲除菌酪氨酸产量进一步提高至44.5 g/L和35.1 g/L,较出发菌株酪氨酸产量分别提高了57%和24%。研究结果对代谢工程强化大肠杆菌生产酪氨酸具有重要的参考价值。     

大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控.利用Red重组技术,在mtr单基因敲除菌的基础上,成功构建了mtr.tnaB和mtr.aroP双基因敲除菌以及mtr.tnaB.aroP三基因敲除菌,并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响.发酵结果表明,mtr.tnaB和mtr.aroP双基因缺失后,色氨酸产量分别达到1.38 g/L和1.27 g/L,与出发菌株相比分别提高了17％和9％,而mtr.tnaB.aroP三基因缺失后,菌体生长受到了明显抑制,发酵后色氨酸产量仅为0.63 g/L.在补料分批发酵实验中,mtr.tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L,与出发菌株相比色氨酸产量提高了27％.     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims:  The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water.
Methods and Results:  Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r  = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci.
Conclusions:  The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria.
Significance and Impact of the Study:  The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.     

A plasmid-encoded arsenite pump produces arsenite resistance in Escherichia coli

The arsenate resistance operon of R-factor R773, a conjugative resistance plasmid, has two functional regions, a promoter-proximal region encoding resistance to arsenite and antimonate, and a promoter-distal one encoding arsenate resistance. Cells bearing arsenite resistance plasmids exhibited reduced accumulation of 74AsO2-. When resistant cells were depleted of endogenous energy reserves and then loaded with 74AsO2-, active extrusion of the ion was observed when an energy source was supplied. Intracellular ATP was required for extrusion, but a proton motive force was neither necessary nor sufficient. An arsenite-sensitive mutant was unable to extrude arsenite, while an arsenate-sensitive mutant had normal arsenite transport. These results suggest that the action of a plasmid-encoded primary arsenite efflux pump is the mechanism of arsenite resistance.     

Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method

Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

基因敲除提高大肠杆菌工程菌生产异丁醇产量的研究

探究pflB、frdAB、fnr和AdhE四基因缺失突变株对大肠杆菌工程菌发酵生产异丁醇的影响。运用Red重组系统敲除大肠杆菌BW25113的pflB、frdAB、fnr和AdhE基因,构建pflB、frdAB、fnr和AdhE四基因缺失突变株E.coliBW25113H,结合本实验室已经构建的表达质粒pSTV29-alsS-ilvC-ilvD-kdcA,并检测该工程菌在1L发酵罐的发酵过程中的生物量、突变菌株的稳定性、异丁醇产量及有机酸含量的变化情况。成功获得pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H。发酵结果表明,该工程菌能以较长时间,较高比生长速率保持对数生长期,其稳定性较好,异丁醇产量增加了40%。成功构建pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H,结合非自身发酵途径使异丁醇的产量由3 g/L提升至4.2 g/L。     

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18

In this study, Agilent two‐colour microarray‐based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full‐sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two‐fold change minimum threshold, we found 18 genes that were differentially expressed (10 up‐regulated and eight down‐regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation‐ and immune‐related pathways. Based on the genes identified in the screen and the pathway analysis results, real‐time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid‐related pathways), SLA‐1 and SLA‐3 genes (of inflammation‐ and immune‐related pathways), as well as the differential genes FUT1, TAP1 and SLA‐DQA. Subsequently, real‐time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.     

Decreased cupric ion uptake as the mechanism for cupric ion resistance in Escherichia coli

Abstract Plasmid-encoded copper (Cu²⁺) resistance in Escherichia coli was due to decreased uptake of Cu²⁺. The Cu²⁺-resistant E. coli Rtsl strain contained a 60 MDa plasmid which is known to encode for both Cu²⁺ and kanamycin resistance. A plasmid-free derivative of the same organism exhibited a greater uptake of Cu²⁺, and sensitivity to Cu²⁺ in both respiration and growth studies than the E. coli Rtsl strain.