清华大学生物工程试题预测!(某研究生gg版)

生物工程预测！（某研究生gg版）国强：必考（只考？）商业运作计划。

考且只考。

Industrial Microbiology is the discipline that uses microorganisms, usually grown on a large scale, to produce valuable commercial products or carry out important chemical transformations. Fermentation Technology is the technology to grow cells in a large scale with high efficiency, it also includes product recovery processes. Flowsheet（流程）for developing an industrial microbial fermentation process：◈1 Strain selection 菌种筛选–Purchase from Culture Collections从菌种收藏中心购买–Screening of nature circumstances 从自然界中筛选–Genetic engineering IIncorporation into artificial plasmids of genes from a wide variety of sources has made possible the transfer of genetic material across virtually any species barrier–Mutations–Cell biology techniquesProtoplast fusion (promote high frequencies of genetic recombinants)◈2 Laboratory process developmentShake flash ExperimentsLab scale fermentor (5-10 L)Pilot scale fermentor (300-3000 L)Commercial fermentor (10,000-500,000 L)Shake Flask ExperimentsOptimization of conditions for cell growth and product formation using shake flask experiments:1. pH2. Temperature3. Dissolved oxygen (DO)4. Substrate choice5.Maximal and optimal substrate concentration6. OthersFermentor Experiments1. Agitation2. Cooling and heating3. Air inlet and outlet4. pH control5. Nutrient addition6. Inoculation7. Viewing portLab scale fermentor ExperimentsBatch process：All the nutrients needed for cell growth will only be added once at the beginning of fermentation. Fed-batch process：During the fermentation, additional nutrients will be added in a batch way to promote the cell growth or product formation and to avoid nutrient deficiency.Continuous process：Semi-continuous process：Similar to continuous process, with the addition of nutrients and outflow of fermentation broth in a continuous and batch way.◈3 Pilot Scale up–To test the feasibility of the lab scale fermentation process in a semi-industrial scale–Pilot fermentors normally have a size ranging from 100 L to 10,000 L, depending on the products to be mass produced later.Problems emerging during the scale up1. As the size of the equipment is increased, the surface-volume ratio changes2. Large fermentor has much more volume for a given surface area, it is obviously more difficult to mix the big tank than the small flask3. In scale up studies on aerobic fermentations, oxygen rate in the fermentor is best kept constant as the size of the fermentor is increased.–How to keep DO constant?◇1 Increase stirring rate◇2 Increase air pressure◇3 Use pure oxygen◇4 Increase air inlet◈4 Industrial Scale up：To transfer the pilot scale results into a commercially feasible production setting. Fermentor sizes range from 100 L to 500,000 L, depending on products.◈5 Downstream process development◈6 Product packaging techniques医药、食品的冷冻干燥包装，酵母粉的脱水包装，诸劝彼崮频韧ǔ５氖品包装，加盐包装，化学药品的保存，普通包装等等。

-Steril Packaging Techniques for Medical Applications and Food Preservation-Pyrogen Free and Steril Packaging for Injection Purpose-Freeze Dry Packaging for Foods or Medicines-Dewatered Packaging (such as Dry Yeast)-Normal Food Packaging (such as Na-glutamate)-Salty Packaging-Preservation Chemicals-Ordinary Packaging◈7 Other commercial considerationStrengths、WeaknessesOutline of total capital investment→Fixed capital–Direct costs (Land, site development, buildings, processing, services)–Indirect costs(Engineering,construction, Contingency, Fees)–Start-up costs→Working capital–Inventory, accounts receivable, account payable☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆总体上来说和前年的题比较像，他其实就是想考Ti质粒，不过不好意思像梦梦和国强那样每年都出一样的题吧，对呀，我都不知道是谁讲过的。

小刘：Ti质粒分子机制、rt-pcr（逆转录－PCR）。

★★反向PCR、反向聚合酶链反应：用适当的限制性内切酶裂解含核心区的DNA，以产生适合于PCR扩增大小的片段，然后片段的末端再连接形成环状分子，从而将边侧区域转化为内部区域。

PCR的引物同源于环上核心区的末端序列，但其3'端趋向未知区域，其方向性使链的延长经过环上的未知区而不是分开引物的核心区。

典型的PCR扩增使用与互补链杂交的寡聚核苷酸引物。

引物是定向的，使延伸向内跨过两个引物之间的区域。

一个引物的DNA合成产物作为另一个引物的模板，进行DNA变性、引物退火，DNA聚合酶管伸反应的多次重复性循环，可使引物规定区域的拷贝数成指数增加。

但用传统PCR方法得不到紧邻引物外侧的DNA序列，因为寡聚核苷酸所引导的既有目的DNA又有引物外侧区的DNA合成在拷贝过程中只呈线性增长，这种线性增长是因为，对于每种引物来讲，其不能引导DNA反向合成(3'-5').反向PCR的应用：边侧区域的扩增；末端特异探针的制备；序列的易位、转座和基因融合；测定未知边侧序列。

主要优点是简单快速，可以研究许多独立克隆。

其某些应用适合于临床诊断。

局限：一是由未知边侧序列性质引起的，需用几种酶试验以选择产生大小合适的片段的内切酶；二是许多常用内切酶也在不适当位点裂解载体序列。

但一旦确定合适的内切酶，反向PCR方法是直接了当和可靠的。

思考题（刘进元）★1 棉纤维细胞发育分为哪四个时期？设计出克隆棉纤维细胞特异表达基因(全长)的实验方案?（不考？）★★2 Real-time PCR与普通PCR的异同点？实时荧光定量PCR技术，是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。

相同点：反应原理、反应体系、反应条件和反应步骤基本相同；不同点：荧光PCR不同于其他PCR的地方在于PCR过程中利用荧光染料在光刺激下释放的荧光能量的变化直接反映出PCR扩增产物量的变化，荧光信号变量与扩增产物变量成正比，并通过足够灵敏的自动化仪器实现对荧光的采集和分析以达到对原始模板量定量的目的。