POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

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.     

ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

FLUORESCENT DNA BINDING DYE-BASED APPROACH FOR MEASURING THE GROWTH OF AN ESCHERICHIA COLI LYSINE AUXOTROPH AND QUANTIFYING LYSINE

Microbiological assays for determination of bioavailable lysine appear to have many advantages. However, since the developed assay is based on bacterial growth and considerable optical density (OD) is required to detect distinguishable differences in extent of growth, it can be time consuming. The purpose of this study was to explore the fluorescence as an alternative method to measure bacterial growth instead of OD and examine the possibility to shorten the time required for the lysine assay. An assay based on SYTO 9 green fluorescent DNA binding dye (Live/Dead BacLight Protocol, Molecular Probes) was used to stain all bacteria in a population. Additional experiments were carried out to determine the ability of fluorescence based on SYTO 9 to overcome problems associated with high nonbacterial background that contributes to OD. From this study it appears that using fluorescence based on SYTO 9 green fluorescent staining, the E. coli lysine auxotroph growth assay can be completed in 9 h instead of 11 h and has the advantage of improved detection sensitivity. Problems associated with interference by high background nonbacterial OD can be partially resolved by fluorescence.     

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。     

抗肿瘤蛋白Onconase的原核细胞表达及细胞毒性检测

本文主要利用pET22b(+)表达载体在大肠杆菌中表达一种在两栖类蛙(Rana pipiens)卵细胞内存在的核糖核酸酶—Onconase。通过包涵体复性、蛋白纯化等程序最终获得与天然蛋白活性相似的重组蛋白,并检测Onconase 对源于皮肤T 细胞淋巴瘤的Hut-78肿瘤细胞的毒性(IC_(50)=0.5μmol/L),证明Onconase 用于抗淋巴瘤的可行性。     

白眼家蝇的特性及其在生物测定上的应用

1961年8月,在北京郊区农药厂附近采集了一些家蝇,经过室内培养后,发现有7个白眼家蝇,随即进行分离培养,成为白眼变种品系。 白眼家蝇变种在国内还是第一次发现,国外亦只是近二三年来才有报道(Sullivan及Hiroyoshi,1960;Tsukamoto,1961),他们发现的变种可能属于 Musca domestica domes-tica L.而我们发现的是属于 Musca domestica vicina M.。     

THE ASSOCIATED GROWTH OF STREPTOCOCCUS LACTIS AND ESCHERICHIA COLI

SUMMARY: The possibility that contamination of farmhouse starters by coli-aerogenes bacteria may be a factor in producing the subtle flavour of farmhouse Cheddar cheese has been discussed. The associated growth of Strep. lactis and E. coli I at 30° and 37° resulted in the rapid disappearance of E. coli from the mixtures, even though it had been the dominant organism in some of them originally. Mixtures containing Strep. lactis and an anaerogenic strain of E. coli still contained this variant at the end of a month, although in no definite ratio and in a very much reduced proportion. It is concluded that the components of coli-lactic starters to be used in the manufacture of cheese should be combined together in the vats.     

THE INCREASED POTENTIAL FOR SELECTION OF THE LAC OPERON OF ESCHERICHIA COLI

The fitness effects of six lac operons from natural isolates of Escherichia coli were determined in chemostats, in a test of the idea that selective differences among natural alleles are greater in novel conditions than in the prevailing environment, resulting in a greater genetic variance in fitness in novel conditions. Fitnesses were determined in the common milk sugar lactose, the natural substrate of the lac operon, and in three rare β-galactosides, lactulose, galactosyl-arabinose, and methyl-galactopyranoside, that are novel for E. coli. Significantly greater fitness differences were observed among the lac alleles in each of the novel β-galactosides than in lactose. An alternative explanation of the experimental findings is discussed. General evolutionary causes and consequences of selection potentials are discussed, and an outline of the work necessary to further elucidate the physiological basis of the observed potential for selection of the lac operon of E. coli is presented.     

利用大肠杆菌质粒检测与分离痘苗病毒的启动子

本文发现,痘苗病毒DNA一些巳知的启动子序列和一些功能尚不清楚的DNA片段,可以在大肠杆菌中起始氯霉素乙酰基转移酶(Chloramphenicol Acetyltrsnsferase,简称CAT)基因的转录和表达,使转化细菌呈现氯霉素抗性表型,这一结果证明,痘苗病毒的启动子可以被大肠杆菌的RNA多聚酶所识别并有效工作。同时发现不同启动子具有不同的强度,利用大肠杆菌质粒分离和检测痘苗病毒的启动子序列,不仅可以研究痘苗病毒基因组的表达调节特点,而且也为组建痘苗病毒表达载体提供了一个快速、简便可靠的方法。     

抗性库蚊酯酶基因在大肠杆菌中的克隆和表达

用抗性库蚊酯酶基因,引入原核表达载体pRL439,转化大肠杆菌HB101细胞,获得表达。通过酶切、Southern杂交鉴定重组质粒。研究了重组菌酯酶的活性,重组质粒pRLB1表达的酯酶具有高酶活并能高效降解酯酶的特异性底物α乙酸萘酯(αNA)和β乙酸萘酯(βNA);经对重组菌进行细胞固定化后降解农药三氯杀虫酯(7504),反应时间短,降解效率高     

THE EFFECTS OF SULFATHIAZOLE AND OF PROPYL CARBAMATE ON THE RATE OF OXYGEN CONSUMPTION AND GROWTH IN ESCHERICHIA COLI

1. The rates of growth and of oxygen consumption by cells of E. coli have been measured under identical conditions, and the effects of sulfathiazole (ST) and of n-propyl carbamate (PC) on these two processes have been compared. 2. The rate of growth was measured by (a) the increase in the viable cell count, (b) the increase in the optical density of the culture, (c) the increase in the rate of oxygen consumption, and (d) the decrease in the ammonia of the medium. The results as indicated by these several measures were identical under the conditions of these experiments. 3. Concentrations of ST or of PC which are just sufficient to stop growth completely, lower the rate of oxygen consumption per unit of bacterial protoplasm to a value approximately 50 per cent of that seen in the absence of the inhibitor. 4. It is shown that the rate of oxygen consumption in cells from old cultures is less affected by ST than is the rate of oxygen consumption by cells from young cultures. It is probable that the rate of oxygen consumption by "old" cells is lower than that of "young" cells. 5. The effects of ST and PC on both the rate of oxygen consumption and the rate of growth are very similar, indicating in a general way, that the mechanism of the actions of these two inhibitors is similar. Furthermore, since both of them produce appreciable inhibition of the rate of oxygen consumption while they are inhibiting growth, the possibility that the effect on oxygen consumption is the immediate cause of the effect on growth must be entertained.