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Tyrosine Biosynthesis PowerPoint Presentation
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Tyrosine Biosynthesis

Tyrosine Biosynthesis

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Tyrosine Biosynthesis

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  1. Tyrosine Biosynthesis Yao 05/12/12

  2. Pathway design glk Ptrc Ptrc Plac-UV5 Plac-UV5 ptsG- p15A ori pMB1 ori 6-P-G 6-P-GA aroGfbr tyrB aroA ppsA pckA tyrAfbr aroB aroE T1 T1 T2 T2 tktA aroL evgA glk Phe csrA53:FRT tyrR- tktA E4P pykF- pheA- pykA- aroL aroGfbr aroE aroA aroB PEP CA DAHP tyrAfbr ppsA pyruvate tyrB pckA ppc- Tyr oxaloacetate Gene regulation Gene overexpression Gene deletion Original gene fbr:feedback-inhibition-resistant

  3. Plasmid construction 10~15 copy 15~20 copy

  4. peasyT1-aroGfbr-tyrAfbr-aroE Colony PCR verification Primer up: M13R Primer down:aroG down 1200bp

  5. peasyT1-aroGfbr-tyrAfbr-aroE Enzyme digest HindIII +speI 3600bp+3300bp

  6. pACYC-ppsA-tktA-glk Enzyme digest verification EcoRI +BamHI 4700bp+5400bp Colony PCR verification Primer up: pACYC up Primer down:glk down Theoretical size: 1862 bp

  7. pYMB1-aroGfbr-tyrAfbr-aroE Enzyme digest HindIII +speI 2700bp+3300bp Colony PCR verification Primer up: tyrB down Primer Rev:pYMB down Theoreticalsize: 1400 bp

  8. pYMB1-aroGfbr-tyrAfbr-aroE-glk-tktA-ppsA Colony PCR verification Primer up: pYMB up Primer Rev:ppsA down Theoretical size: 2862 bp

  9. peasyT1-tyrB-pckA Enzyme digest HindIII +speHI 3170bp+2700bp Colony PCR verification Primer up: pckA down Primer Rev:tyrB up Theoretical size: 2870 bp

  10. Summary glk Ptrc Ptrc Plac-UV5 Plac-UV5 ptsG- p15A ori pMB1 ori 6-P-G 6-P-GA aroGfbr tyrB aroA ppsA pckA tyrAfbr aroB aroE T1 T1 T2 T2 tktA aroL evgA glk Phe csrA53:FRT tyrR- tktA E4P pykF- pheA- pykA- aroL aroGfbr aroE aroA aroB PEP CA DAHP tyrAfbr ppsA pyruvate tyrB pckA ppc- Tyr oxaloacetate Gene regulation Gene overexpression Gene deletion Original gene fbr:feedback-inhibition-resistant

  11. Work plan Ptrc Plac-UV5 p15A ori tyrB aroA pckA aroB T1 T2 aroL evgA 做一下pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA的qRT-PCR,看一下各个基因的转录情况。 转化pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA至已经敲除好的菌株中,发酵,测一下产量。 测量转化pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA的菌株中的胞内产物,主要是E4P、PEP、Shikimicacid的胞内含量。

  12. LC-MSmethods 样品淬灭 指数生长后期发酵液 5 mL,20 mL,-40℃ 甘油 /0. 9% NaCl( 60∶ 40,v / v) 溶液混合,12 000 r/min,-19 ℃3 min 去上清液,用 5 mL,4 ℃ ,0. 9% NaCl溶液洗涤菌体,再于12 000 r/min,4 ℃,3 min,弃去上清液,得到新鲜干净的菌体于离心管中,供提取使用。 样品提取 在含有菌体的离心管中加入5 mL 50%甲醇水溶液,漩涡混合混匀,液氮冰冻3 min,冰上解冻5 min,漩涡混合,重复上述液氮冻融过程2 次,15 000 r / min,4 ℃ 离心 5 min,上清液于干净的离心管中,冷冻干燥后,-80 ℃保存,待进样作 LC-MS/MS 分析。 分析条件 色谱柱: Waters Atlantis HILIC silica柱( 150 mm ×2. 1 mm,3 μm,Ireland) ; (AgelaVenusilHILIC 4000) 柱温: 35 ℃; 进样体积: 5 μL; 流速: 250 μL/min; 流动相 A: 10mmol / L 乙酸铵溶液,流动相 B: 乙腈 梯度洗脱程序: 10% A 保持 3 min; 3 ~ 15 min,10% A 线性变化至 50%A 并保持至 25 min; 25 ~27 min 50% A 线性 变化至 10%A 并平衡至 35 min。 MS / MS 条件。电喷雾离子 ( ESI) 源; 负离子扫描; 喷雾电压 -4. 2 kV; 离子源温度 500 ℃; 气帘气压25 psi; 喷雾气压 45 psi; 加热辅助气压 40 psi。多反应监测扫描模式( MRM) : 入口电压 - 5 V,出口电压- 3 V

  13. Thanks Yuanfeng Yao +86 15900318354 yuanfengyao@gmail.com