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     

一种快速精确编辑疱疹病毒基因组的方法

为了快速且准确地对疱疹病毒基因组进行基因敲除、插入或者点突变等修饰,通过同源重组将马立克氏病病毒 (MDV) 超强毒株Md5基因组克隆到细菌人工染色体 (BAC)。将筛选的阳性重组体DNA电转进DH10B菌株,用PCR及限制性片段多态分析 (RFLP) 方法鉴定含Md5全基因组的BAC克隆。将阳性重组体DNA转染入鸡胚成纤维细胞 (CEF),拯救出重组病毒,命名为Md5BAC。进一步利用Red酶介导的两步法基因重组技术构建MDVlorf10基因敲除毒株。为了验证被敲除基因功能的特异性,将lorf10插入原位点以构建基因复原毒株。将构建的重组毒株分别感染CEF细胞,用间接免疫荧光试验确认重组病毒均包装成功;病毒生长曲线结果表明,lorf10敲除不影响病毒的体外增殖。总之,这为其他疱疹病毒的基因组编辑提供了技术参考。     

营养缺陷型酵母的鉴定技术

营养缺陷型酵母的鉴定技术张明（安徽农业大学生物工程系,合肥２３００３６）王元君,潘仁瑞（中国科学技术大学生物系,合肥２３００２６）通过固体法和液体法鉴定酵母营养缺陷型的实验,证明了液体法较固体法精确,并用液体法成功地鉴定了粟酒裂殖酵母（ｓｃｈｉｚｏｓ...     

一种新的基于Red重组及I-Sec I体内切割的大肠杆菌基因组无痕删减方法

目前常用的基因修饰方法是在Red同源重组介导下,电转线性PCR片段替换染色体上指定序列。因PCR过程错误掺入,该方法常常会在同源序列部位产生一些突变。为了避免此类突变,我们建立了一种新的无痕删除方法。首先将含有抗性标记(两侧带有I-Sec I识别位点)的线性DNA电转到Red重组感受态细胞内,用抗性基因替换基因组上指定序列;然后,将携带融合同源臂(两侧带有I-Sec I位点)的供体质粒导入上述细胞,诱导表达I-Sec I内切酶切割供体质粒释放同源片段,同时切除染色体上抗性基因产生双链断裂,通过分子间同源重组实现无痕删除。我们应用该方法连续删除了大肠杆菌DH1基因组上11个非必需区,使基因组减小10.59%。PCR测序证明所有删减区域同源臂未发生突变,基因组重测序证明指定区域被删除。删减菌的生长变化不大,但耐酸能力有所改变,并对番茄红素合成有不同影响。     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究, 我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因, 并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3为标记基因, 在不含尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株, 该菌株在低K+培养基中导入拟南芥K+转运体基因AtKuP1可恢复正常生长。     

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。     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. III. ADAPTATION OF ESCHERICHIA COLI TO A TEMPORALLY VARYING ENVIRONMENT

Six lines of the bacterium Escherichia coli were propagated for 2,000 generations in a temporally varying environment. The imposed environmental regime consisted of alternating days at 32°C and 42°C, with rapid transitions between them. These derived lines are competitively superior to their ancestor in this variable temperature regime. We also measured changes in the fitness of these lines, relative to their common ancestor, in both the constant (32°C and 42°C) and transition (from 32°C to 42°C and from 42°C to 32°C) components of this temporally varying environment, to determine whether the bacteria had adapted to the particular constant temperatures or the transitions between them, or both. The experimentally evolved lines had significantly improved fitness in each of the constant environmental components (32°C and 42°C). However, the experimental lines had not improved in making the sudden temperature transitions that were a potentially important aspect of the temporally variable environment. In fact, fitness in making at least one of the transitions (between 32°C and 42°C) unexpectedly decreased. This reduced adaptation to the abrupt transitions between these temperatures is probably a pleiotropic effect of mutations that were responsible for the increased fitness at the component temperatures. Among the six experimental lines, significant heterogeneity occurred in their adaptation to the constant and transition components of the variable environment.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. IV. ADAPTATION OF ESCHERICHIA COLI AT A NICHE BOUNDARY

Following an environmental change, the course of a population's adaptive evolution may be influenced by environmental factors, such as the degree of marginality of the new environment relative to the organism's potential range, and by genetic factors, including constraints that may have arisen during its past history. Experimental populations of bacteria were used to address these issues in the context of evolutionary adaptation to the thermal environment. Six replicate lines of Escherichia coli (20°C group), founded from a common ancestor, were propagated for 2000 generations at 20°C, a novel temperature that is very near the lower thermal limit at which it can maintain a stable population size in a daily serial transfer (100-fold dilution) regime. Four additional groups (32/20, 37/20, 42/20, and 32–42/20°C groups) of six lines, each with 2000 generation selection histories at different temperatures (32, 37, 42, and daily alternation of 32 and 42°C), were moved to the same 20°C environment and propagated in parallel to ascertain whether selection histories influence the adaptive response in this novel environment. Adaptation was measured by improvement in fitness relative to the common ancestor in direct competition experiments conducted at 20°C. All five groups showed improvement in relative fitness in this environment; the mean fitness of the 20°C group after 2000 generations increased by about 8%. Selection history had no discernible effect on the rate or final magnitude of the fitness responses of the four groups with different histories after 2000 generations. The correlated fitness responses of the 20°C group were measured across the entire thermal niche. There were significant tradeoffs in fitness at higher temperatures; for example, at 40°C the average fitness of the 20°C group was reduced by almost 20% relative to the common ancestor. We also observed a downward shift of 1–2°C in both the upper and lower thermal niche limits for the 20°C selected group. These observations are contrasted with previous observations of a markedly greater rate of adaptation to growth near the upper thermal limit (42°C) and a lack of trade-off in fitness at lower temperatures for lines adapted to that high temperature. The evolutionary implications of this asymmetry are discussed.     

ADAPTATION OF A METHIONINE AUXOTROPH ESCHERICHIA COLI GROWTH ASSAY TO MICROTITER PLATES FOR QUANTITATING METHIONINE

A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. I. FITNESS RESPONSES OF ESCHERICHIA COLI TO CHANGES IN ITS THERMAL ENVIRONMENT

We used bacteria to study experimentally the process of genetic adaptation to environmental temperature. Replicate lines of Escherichia coli, founded from a common ancestor, were propagated for 2,000 generations in 4 different thermal regimes as 4 experimental groups: constant 32, 37, or 42°C (thermal specialists), or a daily alternation between 32 and 42°C (32/42°C: thermal generalists). The ancestor had previously been propagated at 37°C for 2,000 generations. Adaptation of the groups to temperature was measured by improvement in fitness relative to the ancestor, as estimated by competition experiments. All four experimental groups showed improved relative fitness in their own thermal environment (direct response of fitness). However, rates of fitness improvement varied greatly among temperature groups. The 42°C group responded most rapidly and extensively, followed by the 32 and 32/42°C groups, whose fitness improvements were indistinguishable. The 37°C group, which experienced the ancestral temperature, had the slowest and least extensive fitness improvement. The correlated fitness responses of each group, again relative to the common ancestor, were measured over the entire experimental range of temperatures. No necessary tradeoff between direct and correlated responses of fitness was apparent: for example, the improved fitness of the 42°C group at 42°C was not accompanied by a loss of fitness at 37°C or 32°C. However, the direct fitness responses were usually greater than the correlated responses, judged both by comparing direct and correlated responses of a single group at different temperatures and by comparing direct and correlated responses of different groups at a single temperature. These comparisons indicate that the observed adaptation was, in fact, largely temperature specific. Also, the fitness responses of the generalist group across a range of temperatures were less variable than those of the thermal specialist groups considered as whole.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE II. THERMAL NICHES OF EXPERIMENTAL LINES OF ESCHERICHIA COLI

Groups of replicated lines of the bacterium Escherichia coli were propagated for 2,000 generations at constant 32, 37, or 42°C, or in an environment that alternated between 32 and 42°C. Here, we examine the performance of each group across a temperature range of 12-44°C measuring the temperatures over which each line can maintain itself in serial dilution culture (the thermal niche). Thermal niche was not affected by selection history: average lower and upper limits remained about 19 and 42°C for all groups. In addition, no significant differences among groups were observed in rate of extinction at more extreme temperatures. Within the thermal niche, we measured the mean fitness of the evolved groups relative to their common ancestor. Increases in mean fitness were temperature specific, with the largest increase for each group occurring near its selected temperature. Thus, the temperature at which mean fitness relative to the ancestor was greatest (the thermal optimum) diverged by about 10°C for the groups selected at constant 32°C versus constant 42°C. Tradeoffs in relative fitness (decrements relative to the ancestor elsewhere within the thermal niche) did not necessarily accompany fitness improvements, although tradeoffs were observed for a few of the lines. We conclude that adaptation in this system was quite temperature specific, but substantial divergence among groups in thermal optima had little effect on the limits of their thermal niches and did not necessarily involve tradeoffs in fitness at other temperatures.     

大肠埃希菌在肿瘤患者肠外的分布及耐药谱分析

目的：了解大肠埃希菌在肿瘤患者肠外的分布和感染情况及耐药性。方法：参照全国临床检验操作规程,采用K-B法对云南省肿瘤医院58例肿瘤患者继发大肠埃希菌感染进行分析及对9种抗生素的耐药谱测定。结果：各类肿瘤患者中,以宫颈癌及继发大肠埃希菌感染多见,其中,宫颈癌为28.30%。肺癌为26.87%。从标本来源来看,以尿液标本最多,为63.79%,其次为痰液12.07%及分泌物12.07%。结论：大肠埃希菌在肿瘤患者肠外分布广泛,所致感染较严重,经耐药谱测定发现大肠埃希菌多重耐药类型多,提示对肿瘤患者治疗应重视局部微生态平衡及控制感染。     

CONSTRUCTION AND GROWTH KINETICS OF A BIOLUMINESCENT METHIONINE AUXOTROPH ESCHERICHIA COLI STRAIN FOR POTENTIAL USE IN A METHIONINE BIOASSAY

Methionine is one of the essential and first limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibits a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methionine quantitation. Transformants were selected based on antibiotic resistance and plasmid containing candidates were confirmed by restriction enzyme digestion and gel electrophoresis. To confirm the bioluminescent phenotype, video imaging of the strain using long exposure photography yielded colonies exhibiting bioluminescence. The strain was also tested in the presence of ampicillin supplemented media with increasing methionine concentrations and growth response (measured as optical density, OD), growth rates and methionine affinities were compared before and after transformation. Although the transformed E. coli methionine auxotroph exhibited somewhat different growth kinetic responses than the nontransformed strain, the standard curves used for estimating methionine concentrations were not different. Based on the results in this study the transformed bioluminescent strain could be used as an OD-based assay if bioluminescence equipment and materials are not available.     

大肠杆菌L型生物学特性及致病性的研究

我们对人工诱导的大肠杆菌L型从生物学特性、致病性以及耐药性R质粒传递等方面进行了初步的研究。结果表明:L型菌从菌体形态、菌落特征、生化反应等方面与原型菌有较大差别;原型大肠杆菌和相应的L型菌接合试验均阳性,但后者接合频率较前者明显低;将L型菌经膀胱和尾静脉感染小鼠,引起实验鼠脏器的间质性炎症,通过病理学和细菌学检查证明,L型菌可直接引起脏器感染,并非只有返祖后才能致病。     

日光辐射对大肠杆菌宿主菌和工程菌存活影响的初步研究

本文了不同辐射强度的自然日光照射下,对大肠杆菌宿主菌和工程菌在光滑表面上的存活影响。结果表明,暴露２０ｍｉｎ日光累积总辐射１．１４ＭＪ／ｍ＾２,宿主菌的存活率为０．０２％。工程菌则为０。散射暴露８０ｍｉｎ,日光累积强度０．９０ＭＪ／宿住菌和工程菌的存活率比值为２：１。黑暗环境下,暴露６天对主菌采样有细菌存活,工程菌暴露２天的存活率为０。