实验一、染色体步移法克隆已知序列的侧翼序列

现代微生物学实验技术

马玉超

北京林业大学生物科学与技术学院办公地点：生物楼117；电话：62336016；

Email:myc0307@

实验内容

♣染色体步移法克隆已知序列的侧翼序列♣转座子随机突变及目的表型的筛选

♣-

大肠杆菌β半乳糖苷酶的诱导

♣利用SDS-PAGE分析融合蛋白的可溶性♣绿色糖单胞菌的发酵及孢外酶的提取

染色体步移法克隆已知序列的侧翼序列

TAIL PCR

—TAIL-PCR

牛伯庆

实验目的

1.掌握TAIL-PCR的原理，增强对PCR重要性的认识；1TAIL PCR的原理增强对重要性的认识

2.掌握TAIL-PCR的实验流程，为将来课题研究奠定

2的实验流程为将来课题研究奠定基础。

特异引物序列：

Sp1:CGAGCAGATGGTTCTTGG

Sp2:TGGCGAGTGAGACGACGAGT

Sp3:TGGAGAACTT GTAACCCTTG GTG

a hot arbitrary degenerate primers: AD1：5´-TG(A/T)GNAG(A/T)ANC(G/C)AGA-3´；

AD2：5AG(A/T)GNAG(A/T)ANCA(A/T)AGG3；

5´-AG(A/T)GNAG(A/T)ANCA(A/T)AGG-3´

AD3：5´-CA(A/T)CGICNGAIA(G/C)GAA-3´；

AD4：5TC(G/C)TICGNACIT(A/T)GGA3。

5´-TC(G/C)TICGNACIT(A/T)GGA-3´

TAIL‐PCR:Thermal asymmetric interlaced PCR

Liu et al., 1994

al

实例：微生物法生产丙烯酰胺

丙烯酰胺

最简单和最重要的酰胺化合物

聚丙烯酰胺：百业助剂

采油水处理造纸其它

微生物法生产丙烯酰胺Nitrile

Hydratase (NHase)

Pseudomonas Bacillus

Rhodococcus

NHase

氮代谢网络

课题背景-腈水合酶代谢酶系研究进展腈水合酶代谢酶系研究进展

微生物法生产丙烯酰胺的菌株鉴定产腈水合酶的菌株Rhodococcus roseus ATCC 271T (X81921)1000.1Scanni electro

48h. B Rhodococcus rhodochrous DSM 43241T (X79288)Rhodococcus gordoniae DSM 44689T (AY233201)Rhodococcus coprophilus DSM 43347T (X80626)Rhodococcus pyridinivorans DSM 44555T (AF173005)Rhodococcus phenolicus DSM 44812T (AM933579)9100845610ing electro

on microgr ars, 5 μ

Rhodococcus rhodnii DSM43336T (X80621)Rhodococcus zopfii ATCC 51349T (X81934)Rhodococcus ruber strain TH Rhodococcus ruber DSM 43338T (X80625)Rhodococcus aurantiacus DSM20162T (X80628)849010052Rhodococcus chlorophenolicus DSM 43826T (X79292)on microg

raph (B) o m Rhodococcus corynebacterioides DSM 20151T (X80615)Rhodococcus terrae DSM 43249T (X53202)Rhodococcus globerulus DSM 43953T (X80620)()Rhodococcus obuensis DSM 43896T (X80634)Rhodococcus bronchialis DSM 43247T (X79287)h d k ()1007810082B graph (A)

of the stra Rhodococcus kyotonensis JCM 23211T (AB269261)Rhodococcus fascians ATCC 12974T (X81930)Rhodococcus erythropolis ATCC19566T (NSU82667)Rhodococcus qingshengii KCTC 19205T (DQ090961)7999100Nocardia asteroides ATCC 19247T (DQ659898)and transm ain TH gro Neighbour-joining tree showing the phylogenetic position of R. ruber strain TH with respect to Rhodococcus species based on 16S rDNA gene sequence. Nocardia asteroides was used as an outgroup.

Numbers at branching points represent bootstrap values from 1000replicates and the cutoff was 50%mission

own on LB Numbers at branching points represent bootstrap values from 1000 replicates, and the cutoff was 50%. Bar, 0.1 substitutions per nucleotide position.

B at 11