基于dCas9和dCpf1构建的遗传基因通路
发布时间：2021-11-10 08:24
正如《自然》杂志所定义的,合成生物学的最终目标是构建有用功能的新型生物系统。为了实现这一目标,我们需要构建新的生物学元件,生物学逻辑门通路和生物学系统。为了成功构建功能型的新型生物系统,生物元件的选择显得尤为重要。CRISPR,规律成簇的间隔短回文重复序列,是细菌和古细菌在长期演化中形成的一种适应性免疫系统,可用来对抗入侵病毒和外界DNA,它具备优秀的可编程性,是合成生物学领域新兴的遗传基因线路构建工具。根据Cas蛋白的不同,可将CRISPR系统分为I型、II型、III型,目前被广泛应用的系统为II型,包括Cas9、Cpf1、C2c2（Cas13a）。在本论文中,主要利用两种CRISPR-Cas II型系统的突变系统去构建遗传基因线路:CRISPR-d Cas9与CRISPR-d Cpf1。这两种突变型系统是将Cas9蛋白与Cpf1蛋白的功能结构域进行突变,使其失去核糖内切酶的功能,但保留它们靶向目的基因的能力,d Cpf1还保留了其加工pre-cr RNA的功能。据已发表的文献描述,当d Cas9蛋白结合到特定DNA元件上,例如,当d Cas9蛋白结合到启动子上游会促进下游蛋白产物的...
【文章来源】：哈尔滨工业大学黑龙江省 211工程院校 985工程院校
【文章页数】：79 页
【学位级别】：硕士
【文章目录】：
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
本文编号：3486925
【文章来源】：哈尔滨工业大学黑龙江省 211工程院校 985工程院校
【文章页数】：79 页
【学位级别】：硕士
【文章目录】：
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
本文编号：3486925
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