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聚酮合成模块组装

聚酮合成模块组装. 林春燕 2010.9.10. Epothilone 56 kb. Erythromycin 33 kb. FIGURE 1 | Synthesis of large DNA molecules in yeast.

august-allison
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聚酮合成模块组装

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  1. 聚酮合成模块组装 林春燕 2010.9.10

  2. Epothilone 56 kb Erythromycin 33 kb

  3. FIGURE 1 | Synthesis of large DNA molecules in yeast. • (a) Yeast homologous recombination mechanism. DNA fragments sharing an overlap region at 3- and5 -ends with the neighboring DNA fragments can be assembled into a single larger DNA molecule. • (b) Construction of a synthetic M. genitalium genome. Twenty-five different overlapping DNA segments (blue arrows, 17–35 kb each) composing the genome were co-transformed into yeast followed by assembly of the entire genome in a single step.

  4. FIGURE 2 | The DNA assembler based strategy for efficient manipulation of natural product biosynthetic pathways. • Various genetic modifications can be introduced in the pathway fragments to be assembled

  5. 29.1 kb • Aureothin金链菌素 • Seven pathway fragments of 4–5 kb, werePCR-amplified from the genomic DNA, and co-transformed with the three helper fragments into S. cerevisiae, leading to their assembly into a single circular DNA molecule of 35.7 kb (29.1 kb from the aureothin biosynthetic pathway and 6.6 kb from the helper fragments).

  6. 45 kb • Spectinabilin • low efficiency (<10%) • most of the constructs underwent severe gene deletion(s) • possibly due to the high sequence identity among PKS domains. • To address this issue, a modified two-step assembly strategy was devised through which we were able to assemble this biosynthetic pathway of 45 kb with an efficiency of 30%.

  7. 54 kb 6-dEB (33kb) DEBS1 DEBS2 DEBS3 M5 M1 TE M3 LD M4 M6 M2 10638 kb (AI) 10704 kb (AII) 9516 kb(AIII) KS AT KR ACP KS AT KR ACP TE KS AT ACP KS AT DH ER KR ACP AT ACP KS AT KR ACP KS AT KR ACP

  8. 6—deb组装方案 • 从糖多孢红霉菌基因组中直接克隆33 kb 6—dEB合成模块。 • 通过PCR克隆八条4~5 kb的片段，与三个辅助片段一起导入酵母中一步组装。若效率过低，改为二步、三步组装策略。 • 化学合成60~80 bp的片段，通过酵母组装系统分别合成KS、AT、KR、ER、ACP、TE，再分别组装合成LD、M1、M2……M6、TE，最后再组装成33 kb目的片段，即全合成思路

  9. 实验计划 • 糖多孢红霉菌接ISP-4平板，传1、2代后，存孢子悬液，并接SM菌丝体培养基，2~3天后提基因组（预计十天内完成）。同时设计引物，送公司合成。 • 以基因组为模板，分别克隆八条4~5 kb片段，使相邻片段约有50 bp同源。同时通过PCR、酶切等方法获得3条辅助片段。 • 将总共十一条片段共同转入酵母中进行组装。

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