實(shí)驗(yàn)一染色體步移法克隆已知序列的側(cè)翼序列FINAL

1、現(xiàn)代微生物學(xué)實(shí)驗(yàn)技術(shù)馬 玉 超辦公地點(diǎn)：生物樓117；電話：；Email:實(shí) 驗(yàn) 內(nèi) 容 染色體步移法克隆已知序列的側(cè)翼序列 轉(zhuǎn)座子隨機(jī)突變及目的表型的篩選 大腸桿菌-半乳糖苷酶的誘導(dǎo) 利用SDS分析融合蛋白的可溶性 綠色糖單胞菌的發(fā)酵及孢外酶的提取 用戶名： 密碼：112233445566染色體步移法克隆已知序列的側(cè)翼序列 TAIL-PCR 牛伯慶 許夢(mèng)秋實(shí)驗(yàn)?zāi)康恼莆誘AIL-PCR的原理，增強(qiáng)對(duì)PCR重要性的認(rèn)識(shí)；掌握TAIL-PCR的實(shí)驗(yàn)流程，為將來(lái)課題研究奠定基礎(chǔ)。a hot arbitrary degenerate primers:AD1：5-TG(A/T)GNAG(A/T)ANC(

2、G/C)AGA-3；AD2：5-AG(A/T)GNAG(A/T)ANCA(A/T)AGG-3；AD3：5-CA(A/T)CGICNGAIA(G/C)GAA-3； AD4：5-TC(G/C)TICGNACIT(A/T)GGA-3。TAIL-PCR 的原理sp1 sp2 sp3已知序列 未知序列 特異引物序列：sp1:TGGAGAACTT GTAACCCTTG GTGsp2:TGGCGAGTGAGACGACGAGTsp3:CGAGCAGATGGTTCTTGG TAIL-PCR:Thermal asymmetric interlaced PCR Liu et al., 1994TAIL-PCR 的原

3、理實(shí)例：微生物法生產(chǎn)丙烯酰胺最簡(jiǎn)單和最重要的酰胺化合物 丙烯酰胺 聚丙烯酰胺：百業(yè)助劑采油水處理造紙其它微生物法生產(chǎn)丙烯酰胺氮代謝網(wǎng)絡(luò)PseudomonasBacillusRhodococcusNHaseNitrile Hydratase (NHase)課題背景-腈水合酶代謝酶系研究進(jìn)展腈水合酶代謝酶系研究進(jìn)展Rhodococcus rhodnii DSM43336T (X80621)Rhodococcus corynebacterioides DSM 20151T (X80615)Rhodococcus rhodochrous DSM 43241T (X79288) Rhodococcus

4、gordoniae DSM 44689T (AY233201) Rhodococcus terrae DSM 43249T (X53202)Rhodococcus globerulus DSM 43953T (X80620)Rhodococcus coprophilus DSM 43347T (X80626)Rhodococcus roseus ATCC 271T (X81921)Rhodococcus pyridinivorans DSM 44555T (AF173005)Rhodococcus phenolicus DSM 44812T (AM933579)Rhodococcus zopf

5、ii ATCC 51349T (X81934)Rhodococcus ruber strain THRhodococcus ruber DSM 43338T (X80625)Rhodococcus aurantiacus DSM20162T (X80628)Rhodococcus obuensis DSM 43896T (X80634)Rhodococcus bronchialis DSM 43247T (X79287)Rhodococcus kyotonensis JCM 23211T (AB269261)Rhodococcus fascians ATCC 12974 T (X81930)R

6、hodococcus erythropolis ATCC19566T (NSU82667)Rhodococcus qingshengii KCTC 19205T (DQ090961)1008499781007984100561001001005282991000.1Rhodococcus chlorophenolicus DSM 43826 T (X79292)Nocardia asteroides ATCC 19247T (DQ659898)微生物法生產(chǎn)丙烯酰胺的菌株鑒定 Neighbour-joining tree showing the phylogenetic position of

7、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 1000 replicates, and the cutoff was 50%. Bar, 0.1 substitutions per nucleotide position.Scanning electron m

8、icrograph (A) and transmission electron micrograph (B) of the strain TH grown on LB at 48h. Bars, 5 m11產(chǎn)腈水合酶的菌株腈代謝相關(guān)基因的克隆與序列分析腈水合酶基因側(cè)翼序列的克隆策略酰胺酶基因側(cè)翼序列克隆的策略6442bpNHase 1869bpSequence the segments of TAIL-PCR2487bpamiE2136bp腈代謝相關(guān)基因的克隆與序列分析9.6kb5.6kb腈水合酶基因簇酰胺酶基因簇RNA聚合酶的亞基組成轉(zhuǎn)錄翻譯表型RNA聚合酶DNAmRNA蛋白質(zhì)全局調(diào)控突變庫(kù)

9、構(gòu)建(Hal Alper, et al. Science. 2006)RNA聚合酶 同時(shí)控制成百上千個(gè)功能基因的轉(zhuǎn)錄； 優(yōu)選突變可以獲得全局最優(yōu)的細(xì)胞表型。Sigma 因子的功能 1 CGCCTCCGCC GACTCGGTGC GCGCCTACCT CAAGCAGATC GGCAAGGTTG CCCTGCTCAA61 CGCCGAGGAG GGGGTCGAGC TCGCCAAGCG CATCGAGGCC GGCCTGTACG CGACCTACCG121 GCTGCAGGAG TACGCCGACA AGGGCGAGAA GCTGCCGGTC GCGCAGCGCC GCGACCTGAA181 CT

10、GGACCTGC CGCGACGGCA ACCGCGCCAA GAACCATCTG CTCGAGGCCA ACCTGCGACT241 CGTCGTCTCA CTCGCCAAGC GCTACACCGG CCGGGGGATG GCCTTCCTGG ATCTCATCCA301 GGAGGGCAAT CTCGGTCTGA TCCGCGCGGT GGAGAAGTTC GACTACACCA AGGGTTACAA361 GTTCTCCACG TACGCCACCT GGTGGATCCG GCAGGCCATC ACCCGCGCCA TGGCGGATCA421 GGCCCGCACC ATCCGCATCC CGGT

11、CCACAT GGTCGAGGTG ATCAACAAGC TCGGCCGCAT481 CCAGCGTGAG CTCCTCCAGG ACCTCGGGCG CGAGCCCACT CCGGAGGAAC TGGCCAAGGA541 GATGGACATC ACGCCGGAGA AGGTACTGGA GATCCAGCAG TACGCCCGGG AGCCGATCTC601 GCTCGACCAG ACCATCGGCG ACGAGGGCGA CAGCCAGCTC GGCGACTTCA TCGAGGACAG661 CGAGGCCGTG GTGGCGGTGG ACGCGGTGTC GTTCACGCTG CTGCAG

12、GATC AGCTGCAGTC721 GGTACTCGAG ACACTGTCCG AACGTGAGGC CGGGGTGGTC CGGCTGCGGT TCGGCCTGAC781 CGACGGTCAG CCGCGGACCC TCGACGAGAT CGGGCAGGTC TACGGGGTCA CCCGCGAGCG841 GATCCGGCAG ATCGAGTCGA AGACCATGTC GAAGCTGCGG CACCTGTCGC GGTCGCAGGT901 GCTGCGCGAC TACCTGGACTGAACGCG部分sigA基因序列紅色紅球菌編碼sigma因子的sigA基因，900bpSp3 Sp2 S

13、p1特異引物序列：sp1:TGGAGAACTT GTAACCCTTG GTGsp2:TGGCGAGTGAGACGACGAGTsp3:CGAGCAGATGGTTCTTGG成 分貯液濃度使用量(50l體系)水12l2GC BufferMg2+ Plus25ldNTPs2.5mmol8l嵌套引物（Sp1）10pmol/l1.5l簡(jiǎn)并引物（AD1、AD2、AD3、AD4）10pmol/l 2l基因組DNA100g/ml1lTaq酶5U/l0.5l操 作 步 驟第一輪TAIL-PCR： 反應(yīng)體系成 分貯液濃度使用量(50l體系)水12l2GC BufferMg2+ Plus25ldNTPs2.5mmol

14、8l嵌套引物（Sp2）10pmol/l1.5l簡(jiǎn)并引物（AD1、AD2、AD3、AD4）10pmol/l 2l第一輪產(chǎn)物(1000倍稀釋)100g/ml1lTaq酶5U/l0.5l操 作 步 驟第二輪TAIL-PCR： 反應(yīng)體系成 分貯液濃度使用量(50l體系)水12l2GC BufferMg2+ Plus25ldNTPs2.5mmol8l嵌套引物（Sp3）10pmol/l1.5l簡(jiǎn)并引物（AD1、AD2、AD3、AD4）10pmol/l 2l第二輪產(chǎn)物 (1000倍稀釋)100g/ml1lTaq酶5U/l0.5l操 作 步 驟第三輪TAIL-PCR： 反應(yīng)體系操 作 步 驟反應(yīng)條件：反應(yīng)循環(huán)

15、次數(shù)反應(yīng)條件第一輪195（2min）594(30s); 60 (1min); 72 (2min30s)194 (30s); 25 (1min); 72 (2min30s)1094 (30s); 44 (1min); 72 (2min30s)1594 (15s); 60 (1min); 72 (2min30s)94 (15s); 60 (1min); 72 (2min30s)94 (15s); 44 (1min); 72 (2min30s)172 (10min)反應(yīng)循環(huán)次數(shù)反應(yīng)條件第二輪1594 (30s); 58 (1min); 72 (2min30s)94 (30s); 58 (1min); 72 (2min30s)94 (30s); 44 (1min); 72 (2min30s)172 (10min)第三輪1594 (