| Generation and Screening of a Non-typeable Haemophilus influenzae Tn-seq Mutant Library
|
|
Author:
Date:
2014-03-05
[Abstract] The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The advantage of using a mariner transposon for the generation of a random transposon mutant library is its insertion into TA sites, which makes the insertion in the genome highly random. In addition, an MmeI restriction site can be introduced in the ...
[摘要] 全基因组筛选Tn-seq（van Opijnen等人，2009）是鉴定具有条件必需功能的细菌基因（例如涉及细菌毒力的基因）的非常有价值的工具。 这些技术基于产生随机突变体文库，其在挑战情况的对照中生长（图1）。 使用水手转座子产生随机转座子突变体文库的优点是其插入TA位点，这使得在基因组中的插入高度随机。 此外，可以在转座子的反向重复中引入MmeI限制位点，而不影响HimarC9转座酶的识别。
|
|
|
| Acetyl-coenzyme A Synthetase (Acs) Assay
|
|
Author:
Date:
2012-09-05
[Abstract] Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represented in nature from bacteria to human. Its function is important for cellular catabolism, especially in order to support microbial growth at low concentrations of acetate (<10 mm)="" (castano="" cerezo="">et al., 2011; Castano Cerezo et al., 2009；Renilla et al., 2012). In this protocol, a continuous coupled enzymatic assay for Acs activity is described. Product formation is followed spectrophotometrically by the formation of NADH. The protocol is tailored for E. coli ...
[摘要] 乙酰辅酶A合成酶（Acs，E.C.6.2.1.1）是从细菌到人广泛代表的乙酸活化酶。其功能对于细胞分解代谢是重要的，特别是为了支持在低浓度的乙酸盐（<10mm）下的微生物生长（castano cerezo等人，2011;="" castano="" cerezo等人，="" ，2009;="">
首先由Brown等人描述了乙酰辅酶A合成酶（Acs）测定法 。 （1977）。使用与苹果酸脱氢酶（Mdh）和柠檬酸合酶（Cs）偶联的酶促方法测量Acs活性：乙酸+ AcASH + ATP→乙酰-CoA + AMP（Cs）乙酰 - CoA +草酰乙酸盐→柠檬酸盐+ Co Wash
（Mdh）L-苹果酸盐+ NAD +→草酰乙酸盐+ NADH
净反应：乙酸盐+ ATP + L-苹果酸盐+ NAD +→柠檬酸盐+ AMP + NADH />在测定条件下，Mdh和Cs活性过量，NADH形成速率受Acs活性限制。
|
|
|