正如《自然》雜志所定義的,合成生物學的最終目標是構建有用功能的新型生物系統(tǒng)。為了實現(xiàn)這一目標,我們需要構建新的生物學元件,生物學邏輯門通路和生物學系統(tǒng)。為了成功構建功能型的新型生物系統(tǒng),生物元件的選擇顯得尤為重要。CRISPR,規(guī)律成簇的間隔短回文重復序列,是細菌和古細菌在長期演化中形成的一種適應性免疫系統(tǒng),可用來對抗入侵病毒和外界DNA,它具備優(yōu)秀的可編程性,是合成生物學領域新興的遺傳基因線路構建工具。根據(jù)Cas蛋白的不同,可將CRISPR系統(tǒng)分為I型、II型、III型,目前被廣泛應用的系統(tǒng)為II型,包括Cas9、Cpf1、C2c2（Cas13a）。在本論文中,主要利用兩種CRISPR-Cas II型系統(tǒng)的突變系統(tǒng)去構建遺傳基因線路:CRISPR-d Cas9與CRISPR-d Cpf1。這兩種突變型系統(tǒng)是將Cas9蛋白與Cpf1蛋白的功能結構域進行突變,使其失去核糖內切酶的功能,但保留它們靶向目的基因的能力,d Cpf1還保留了其加工pre-cr RNA的功能。據(jù)已發(fā)表的文獻描述,當d Cas9蛋白結合到特定DNA元件上,例如,當d Cas9蛋白結合到啟動子上游會促進下游蛋白產(chǎn)物的... 

【文章來源】：哈爾濱工業(yè)大學黑龍江省 211工程院校 985工程院校

【文章頁數(shù)】：79 頁

【學位級別】：碩士

【文章目錄】：
Abstract (In Chinese)
Abstract (In English)
Chapter 1 Introduction
    1.1 The background and significance of the study
    1.2 Research status at home and abroad
    1.3 Brief introduction of synthetic biological circuits mentioned in this paper
    1.4 Main research content and goal
        1.4.1 CRISPR-d Cas9 based genetic circuits
        1.4.2 CRISPR-d Cpf1 based genetic circuits
    1.5 Technology roadmap
Chapter 2 Experiment materials and methods
    2.1 Laboratory equipments
    2.2 Reagents and materials
    2.3 Experimental methods
        2.3.1 Bacterial transformation
        2.3.2 Colony picking
        2.3.3 Mini preparation
        2.3.4 Restriction digest
        2.3.5 Gel electrophoresis
        2.3.6 DNA elution
        2.3.7 Gibson Assembly (One-step isothermal DNA assembly)
        2.3.8 Yeast transformation
        2.3.9 Yeast cells streaking and culture
        2.3.10 Yeast cells induction
        2.3.11 FACS
        2.3.12 RNA extraction
        2.3.13 qRT PCR
Chapter 3 d Cas9-based genetic circuits
    3.1 The design for g RNAs
    3.2 Building d Cas9-based NOT gate
    3.3 d Cas9-based buffer (YES) gate
    3.4 Metabolic burden
    3.5 d Cas9-based beta-estradiol biosensor
    3.6 Discussion
    3.7 Brief summary
Chapter 4 d Cpf1 based genetic circuits
    4.1 Designing a pre-cr RNA tail for the y EGFP m RNA
    4.2 d Cpf1 targeting modified m RNA
    4.3 The construction of d Cpf1 based (galactose) NOT gates
    4.4 Discussion
    4.5 Brief summary
Conclusions
Outlook
References
Papers published in the period of master education
Acknowledgements



本文編號：3486925