全基因组CRISPR干扰筛选和探索内分泌激素对结核分枝杆菌的影响
发布时间：2022-01-13 17:08
结核分枝杆菌（M.tuberculosis）是一种胞内寄生菌,全世界约有三分之一的人口感染M.tuberculosis,但是,大多数人在肺中无害地携带细菌,而没有任何临床症状,被称为潜伏性结核病（LTB）。目前,LTB在全球范围内广泛活化和传播,且尚不清楚其清除的具体机制。此外,结核分枝杆菌对目前用于治疗结核病的抗生素有极大的耐药性,这为结核分枝杆菌感染和在人群中的传播提供了基础。此外,没有良好的诊断工具可以及早发现该疾病。目前可用的结核病疫苗仅对儿童有效。要了解M.tuberculosis的抗药性、毒力及再活化的机制并开发有效的抗结核药物和疫苗,人们应该了解M.tuberculosis的功能基因组学。但是,M.tuberculosis的功能基因组学研究受到复杂且耗时的遗传操作技术的阻碍。基于以上问题,本课题主要集中在如下两个方面进行研究:（1）我们对M.tuberculosis内源性III-A型CRISPR-Cas10系统进行了重新编辑,仅仅通过转入带有mini-CRISPR序列的质粒来简单有效地进行基因编辑、RNA干扰和全基因组CRISPR干扰（CRISPRi）的筛选,同时避免了引...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：109 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
1. Literature review and introduction
1.1 Tuberculosis
1.2 Latent TB
1.3 Epidemiology of TB
1.4 Diagnosis of TB
1.5 Treatment of TB
1.6 Control and Prevention of TB
1.7 Mycobacterium tuberculosis (M. tuberculosis) the causative agent of TB
1.8 Functional Genomics of M. tuberculosis
1.9 Genome complexity and diversity
1.10 Genome editing In M. tuberculosis
1.11 Genome editing In M. tuberculosis
1.12 The CRISPR system
1.13 Effects of stress hormones on M. tuberculosis
2. Aims and Objectives
3. Material and Methods
3.1 Chemicals and reagents
3.2 Bacterial strains, Cell lines and Culture conditions
3.3 Competence cell preparation and electro transformation
3.4 Bacteria treatment and in-vitro growth analysis
3.5 Plasmid construction and Cloning
3.6 DNA Isolation, PCR amplification, purification and plasmid isolation
3.7 RNA extraction and quantitative real-time PCR
3.8 g RNA designing for single gene knockdown
3.9 g RNA library designing and construction
3.10 Library screening and high throughput sequencing
3.11 High throughput sequencing data analysis
3.12 THP-1Cell infection and Treatment with hormones
3.13 Isolation and quantification of Intracellular bacteria
3.14 Other Insilco studies
3.15 Molecular docking
3.16 Statistical analysis
3.17 Reagents Preparation
3.17.1 PBS preparation
3.17.2 LB medium Preparation
3.17.3 7H9broth 7H10 preparation
3.18. Other Protocols
3.18.1 Mt.b DNA Extraction
3.18.2 Protocol for g RNA strands Annealing
3.18.3 Anneal in a thermo cycler using the following parameters
3.18.4 Bbs I Digestion of p MV-261
3.18.5 BbsI Digestion of p MV-261
3.18.6 Ligation reaction
3.18.7 Different hormones preparation
4. Results
Part I
4.1 Genome-wide CRISPR interference screening in M. tuberculosis
4.2 Discussion
Part II
4.3. Elucidation the response of M. tuberculosis toward host stress hormones
4.4. Discussion
5. Conclusion
References
ACKNOWLEDGEMENTS
Supplementary Materials
【参考文献】：
期刊论文
[1]细菌中CRISPR/Cas系统的应用和优化[J]. 傅俊豪,杨发誉,谢海华,谷峰. 生物工程学报. 2019(03)
[2]Advances in the development of molecular genetic tools for Mycobacterium tuberculosis[J]. Chiranjibi Chhotaray,Yaoju Tan,Julius Mugweru,Md Mahmudul Islam,H.M.Adnan Hameed,Shuai Wang,Zhili Lu,Changwei Wang,Xinjie Li,Shouyong Tan,Jianxiong Liu,Tianyu Zhang. Journal of Genetics and Genomics. 2018(06)
本文编号：3586825
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：109 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
1. Literature review and introduction
1.1 Tuberculosis
1.2 Latent TB
1.3 Epidemiology of TB
1.4 Diagnosis of TB
1.5 Treatment of TB
1.6 Control and Prevention of TB
1.7 Mycobacterium tuberculosis (M. tuberculosis) the causative agent of TB
1.8 Functional Genomics of M. tuberculosis
1.9 Genome complexity and diversity
1.10 Genome editing In M. tuberculosis
1.11 Genome editing In M. tuberculosis
1.12 The CRISPR system
1.13 Effects of stress hormones on M. tuberculosis
2. Aims and Objectives
3. Material and Methods
3.1 Chemicals and reagents
3.2 Bacterial strains, Cell lines and Culture conditions
3.3 Competence cell preparation and electro transformation
3.4 Bacteria treatment and in-vitro growth analysis
3.5 Plasmid construction and Cloning
3.6 DNA Isolation, PCR amplification, purification and plasmid isolation
3.7 RNA extraction and quantitative real-time PCR
3.8 g RNA designing for single gene knockdown
3.9 g RNA library designing and construction
3.10 Library screening and high throughput sequencing
3.11 High throughput sequencing data analysis
3.12 THP-1Cell infection and Treatment with hormones
3.13 Isolation and quantification of Intracellular bacteria
3.14 Other Insilco studies
3.15 Molecular docking
3.16 Statistical analysis
3.17 Reagents Preparation
3.17.1 PBS preparation
3.17.2 LB medium Preparation
3.17.3 7H9broth 7H10 preparation
3.18. Other Protocols
3.18.1 Mt.b DNA Extraction
3.18.2 Protocol for g RNA strands Annealing
3.18.3 Anneal in a thermo cycler using the following parameters
3.18.4 Bbs I Digestion of p MV-261
3.18.5 BbsI Digestion of p MV-261
3.18.6 Ligation reaction
3.18.7 Different hormones preparation
4. Results
Part I
4.1 Genome-wide CRISPR interference screening in M. tuberculosis
4.2 Discussion
Part II
4.3. Elucidation the response of M. tuberculosis toward host stress hormones
4.4. Discussion
5. Conclusion
References
ACKNOWLEDGEMENTS
Supplementary Materials
【参考文献】：
期刊论文
[1]细菌中CRISPR/Cas系统的应用和优化[J]. 傅俊豪,杨发誉,谢海华,谷峰. 生物工程学报. 2019(03)
[2]Advances in the development of molecular genetic tools for Mycobacterium tuberculosis[J]. Chiranjibi Chhotaray,Yaoju Tan,Julius Mugweru,Md Mahmudul Islam,H.M.Adnan Hameed,Shuai Wang,Zhili Lu,Changwei Wang,Xinjie Li,Shouyong Tan,Jianxiong Liu,Tianyu Zhang. Journal of Genetics and Genomics. 2018(06)
本文编号：3586825
本文链接：https://www.wllwen.com/projectlw/swxlw/3586825.html
教材专著
