【正文】 費(fèi)曼 Synthetic Biology Why do we need it? Cells are the ultimate Chemical Factory. Synthetic Biology 1 Biology is hierarchical Is it achievable? Synthetic Biology 2 Biology is Modular Is it achievable? Synthetic Biology Hierarchy and Modular (recurrent) anization allows biology to be understandable and synthetic biology to be possible. Is it achievable? Synthetic Biology A possible hierarchy for synthetic biology Synthetic Biology Biological Components: 1Parts Synthetic Biology Biological Components: 2Devices Synthetic Biology Biological Components: 3Systems or Modules Synthetic Biology Biological Components: 3Systems or Modules Basu et al (2023) Nature, 434: 11304 Synthetic Biology Biological Components: 3Systems or Modules Synthetic Biology Biological Components: 3Systems or Modules Synthetic Biology For synthetic biology to bee a form of engineering it will be necessary to achieve precision and reliability. Factors preventing this: 1 Inplete knowledge of biology. 2 Inherent functional overlap (parts with many some unknown functions, some of which are detrimental to the goal in mind. 3 Inpatibility between parts. 4 Parts functionality depends on context. Synthetic Biology as Engineering 2 CI represses expression of unrelated host genes 3 LuxR interacts with CI and blocks its function 4 GFP is nonfluorescent in host Synthetic Biology Synthetic Biology as Engineering Standard Parts Parts should not have multiple functions (One subunit of T7 phage DNA polymerase is actually E. coli thioredoxin) Parts should not encode multiple functions Synthetic Biology Synthetic Biology as Engineering Standard Parts Different parts should be patible Parts should work in different contexts Synthetic Biology Synthetic Biology as Engineering Standard Parts Standardized parts could be easily exchanged between different devices (as well as between different laboratories) Synthetic Biology Synthetic Biology as Engineering Abstraction DNA TGCATGCTGATATACGGCTCGAT Parts Devices Systems Yeast Cloning Sergio Peisajovich Lim Lab June 2023 Experimental Lab Why Yeast? The yeast Saccharomyces cerevisiae (also called ―baker’s yeast‖) is probably the ideal eukaryotic microanism for biological studies. Yeast genome: fully sequenced and easy to manipulate. Basic mechanisms of yeast cell biology (such as DNA replication, rebination, cell division and metabolism) are highly similar to that of higher anisms (including humans). Experimental Lab Yeast Life Cycle Experimental Lab Yeast: Ideal Platform for Synthetic Biology ? Add parts, devices or even modules (in an ―extragenomic‖ format plasmidbased or ―integrating‖ them within the yeast genome. ? Delete specific yeast genes, to remove ―background‖ or interference. ? Add ―reporter genes‖ to monitor in real time the function of the synthetic parts/devices/modules under study. ? Life cycle fast enough so that we could do all these geic manipulations in a reasonable amount of time. Parts/Devices/Modules are built in bacteria Empty initial plasmid Plasmid coding the desired device Transform into Yeast Experimental Lab Yeast: Adding parts… in plasmids Experimental Lab Yeast: Adding parts… in plasmids growth in selective medium Experimental Lab Yeast: Adding parts… into the genome Homologous rebination allows genomic integration, but we still need to select: Experimental Lab Yeast: Adding parts… into the genome Part/Device/Module URA3 plasmid Digest with specific restriction enzyme Part/Device/Module plasmid Linear DNA, ready for yeast transformation and integration Part/Device/Module URA3* Homologous Rebination Yeast Chromosome Ining Linear DNA URA3* URA3 Part/Device/Module Integration (Note that 2 copies, one defective and one functional, of the marker are generated) Yeast Chromosome Experim。不能理解的東西，我無法創(chuàng)造。 DNA breaks Adaptation: Fix it! Side benefit: extreme radiation resistance [D. Radiodurans: incredible resistance] Other extremophiles Desert Varnish – exists in the driest places on Earth Varnish includes bacteria that: ? Arrange clay and manganese above them to shield them from the elements。, and PCR at 72176。) Steps 1 to 5 involve multiple rounds of PCR (heating to 95176。 用途：生物能源、生物除污 … ? Venter下一步的計(jì)劃就是合成某種海藻基因組，這種新型海藻可以通過光合作用把空氣中的二氧化碳轉(zhuǎn)化成汽油或者柴油等清潔能源，從而有效解決目前的氣候變化和能源危機(jī)。 ? 移植： – Venter等把這個合成基因組移植到不含限制性酶切系統(tǒng)的山羊支原體中，基因組能使用后者的酶系統(tǒng)進(jìn)行自我復(fù)制，經(jīng)過多代繁殖后，長成的菌落已經(jīng)純粹由蕈狀支原體組成。 – Venter用化學(xué)方法合成了一千多個約 1kb的 DNA片段，作為這次組裝的基本材料。 – 用計(jì)算機(jī)精確計(jì)算需要合成 DNA分子序列，并用化學(xué)方法合成 A、 T、 G、 C堿基，并使其按所要求序列延伸。 三個步驟：合成、組裝和移植 ? 合成 ： – 蕈狀支原體的基因組是一條大片段的 DNA分子，序列是A、 T、 G、 C四種脫氧核糖核苷酸的排列組合。 ? 此后，他們選擇生長速度更快的蕈狀支原體來做實(shí)驗(yàn)。 – Venter早在 1995年就對生殖支原體測序，并致力于研究維持自由生命的最小基因組。 – 支原體是已知的可以自由生活的最小生物 ,也是最小的原核細(xì)胞。 ? 這也是今年這篇科研論文的雛形，在國外的科學(xué)媒體上曾經(jīng)引發(fā)熱烈的討論。穆尼說，文特爾制造出了“一個基架，在此基架上人們幾乎可以制造出任何東西”，“它可以用于研究新型藥物，也可以用于對人類產(chǎn)生巨大威脅的生物武器”。 ? 然而制造永久生命形式的前景極具爭議性，有可能激起道德、倫理等方面的激烈辯論。” – 他希望這項(xiàng)突破有助于發(fā)展新能源，應(yīng)對氣候變化造成的負(fù)面影響。但這種給特定基因賦予特定任務(wù)的觀點(diǎn)已被眾多生物學(xué)家廣泛接受。由于新的生物體是在現(xiàn)存生物體上搭建，其繁殖和新陳代謝仍然依賴原來生物體的胞內(nèi)機(jī)制。 ? 這種新單細(xì)胞生物體被命名為“合成器”，受 381個基因控制，包含56萬個堿基對。 ? 文特爾研究小組研制出的這種新型染色體即實(shí)驗(yàn)室合成支原體(Mycoplasma laboratorium)，是一種經(jīng)過簡化拼接的生殖支原體(Mycoplasma genitalium)DNA序列，他們將這種合成支原體移植到活細(xì)胞中，使之在細(xì)胞中起主控作用，變換成一種新的染色體。史密斯領(lǐng)導(dǎo)的研究小組在這方面已經(jīng)進(jìn)行了 5年研究。 ? 該研究部分由美國能源部出資，希望藉此研制出新型環(huán)保燃料。 – 用 Mycoplasma mycoides的基因組取代與之關(guān)系密切的 Mycoplasma capricolum的基因組 – C. Lartigue et al. Genome transplantation in bacteria: Changing one species to another Science, June 28, 2023. 人類歷史上第一個人造染色體合成成功 ? 美科學(xué)家稱“人造生命”技術(shù)已被掌握 ? 最具爭議的美國著名科學(xué)家克雷格 J. Craig Venter：基因組替換 ? 成功利用基因組取代技術(shù)，將一種細(xì)菌改變?yōu)榱硪环N