Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate asse...Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate assembly technique,utilizing Escherichia coli as the model organism.Through polymerase chain reaction(PCR)amplification,restriction enzyme digestion,ligation,transformation,and Sanger sequencing,we assessed the operational efficiency and cloning fidelity of both strategies.Our results demonstrated that Golden Gate assembly,leveraging type IIS restriction enzymes and simultaneous ligation,significantly enhanced cloning efficiency and precision,particularly for seamless multi-fragment assembly.In contrast,the classical cloning approach maintained certain advantages in simplicity and robustness for specific experimental conditions.Challenges encountered during transformation and sequencing highlighted the critical impact of technical accuracy on experimental outcomes.This study underscores the importance of selecting appropriate cloning methodologies tailored to experimental objectives and laboratory capabilities,providing a foundation for optimized molecular cloning workflows in future synthetic biology and biotechnology applications.展开更多
程序性死亡受体1(programmed cell death protein 1,PD-1)和程序性死亡配体1(programmed cell death 1 ligand 1,PD-L1)是一对经典免疫检查点,其相互作用在肿瘤免疫逃逸中发挥重要作用。但由于PD-1和PD-L1存在复杂的糖基化修饰,且真核...程序性死亡受体1(programmed cell death protein 1,PD-1)和程序性死亡配体1(programmed cell death 1 ligand 1,PD-L1)是一对经典免疫检查点,其相互作用在肿瘤免疫逃逸中发挥重要作用。但由于PD-1和PD-L1存在复杂的糖基化修饰,且真核细胞中的PD-1和PD-L1在蛋白质水平的表达量低,限制了靶向二者相互作用的药物筛选及抑制剂的开发。本研究针对二者相互作用的关键结构域,利用金门桥组装(golden gate assembly)无缝克隆技术构建了表达功能性截短体蛋白质PD-1^(33-150)和PD-L1^(19-239)的真核载体,并利用HEK-293T细胞系验证了其高效表达。经亲和层析纯化,每升细胞培养基可获得蛋白质纯度>95%的PD-1^(33-150)和PD-L1^(19-239)蛋白分别为5 mg和3 mg。利用生物膜干涉技术(biolayer interferometry,BLI)和流式细胞术(flow cytometry,FCM),我们检测了纯化后PD-1^(33-150)和PD-L1^(19-239)的结合动力学参数、平衡常数以及其细胞结合活性。与昆虫细胞和大肠杆菌在蛋白质水平表达的PD-1胞外域相比,哺乳细胞表达的PD-1^(33-150)对PD-L1的亲和力提高了近24和50倍,同时使结合的解离速率降低至原来的1/400以下,这可能是由于不同的糖基化修饰对PD-1和PD-L1蛋白的相互作用具有重要影响。综上,本研究建立了人源PD-1^(33-150)/PD-L1^(19-239)功能性截短体在蛋白质水平的高效表达与纯化平台,为PD-1/PD-L1抗体药物筛选及小分子免疫检查点抑制剂的开发提供了高质量的分子工具。展开更多
Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TA...Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent （USER）, an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases （TALENs） generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tetl locus, and proved that all can specifically target Tetl gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.展开更多
The assessment of the policy framework governing Invasive Alien Species(IAS) control is of critical importance in conservation. The undertaking of a SWOT analysis of such a framework is necessary because it enhances t...The assessment of the policy framework governing Invasive Alien Species(IAS) control is of critical importance in conservation. The undertaking of a SWOT analysis of such a framework is necessary because it enhances the efficacy of IAS control. The aim of this study is to carry out a SWOT analysis of the policy framework guiding the control of the spreading of Acacia mearnsii and other IAS in the Golden Gate Highlands National Park in South Africa,as well as the implementation of restorative measures in the park and adjacent communities in line with the recommendations of the Convention on Biodiversity. A comparative analysis of this framework and the hierarchical framework that was developed during the European Union Conference on Freshwater Invasives- Networking for Strategy in 2013 is undertaken. The results indicate the need to strengthen parkcommunity relations,upgrade existing legislation,and boost the technical capacity of South African national parks to identify,detect,monitor and predict IAS invasions,both within the parks and their surroundings. This knowledge is important for developing future policies on IAS control in South Africa.展开更多
文摘Molecular cloning remains a cornerstone technique in genetic engineering and synthetic biology.In this study,we conducted a systematic comparative analysis between the classical cloning method and the Golden Gate assembly technique,utilizing Escherichia coli as the model organism.Through polymerase chain reaction(PCR)amplification,restriction enzyme digestion,ligation,transformation,and Sanger sequencing,we assessed the operational efficiency and cloning fidelity of both strategies.Our results demonstrated that Golden Gate assembly,leveraging type IIS restriction enzymes and simultaneous ligation,significantly enhanced cloning efficiency and precision,particularly for seamless multi-fragment assembly.In contrast,the classical cloning approach maintained certain advantages in simplicity and robustness for specific experimental conditions.Challenges encountered during transformation and sequencing highlighted the critical impact of technical accuracy on experimental outcomes.This study underscores the importance of selecting appropriate cloning methodologies tailored to experimental objectives and laboratory capabilities,providing a foundation for optimized molecular cloning workflows in future synthetic biology and biotechnology applications.
基金supported by the National Natural Science Foundation of China (No. Y211291131)the "Strategic Priority Research Program" of the Chinese Academy of Sciences (No. XDA01040109)
文摘Transcription activator-like effectors （TALEs） that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent （USER）, an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases （TALENs） generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tetl locus, and proved that all can specifically target Tetl gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.
基金the Afromontane Research Unit (ARU) for providing financial support for this study
文摘The assessment of the policy framework governing Invasive Alien Species(IAS) control is of critical importance in conservation. The undertaking of a SWOT analysis of such a framework is necessary because it enhances the efficacy of IAS control. The aim of this study is to carry out a SWOT analysis of the policy framework guiding the control of the spreading of Acacia mearnsii and other IAS in the Golden Gate Highlands National Park in South Africa,as well as the implementation of restorative measures in the park and adjacent communities in line with the recommendations of the Convention on Biodiversity. A comparative analysis of this framework and the hierarchical framework that was developed during the European Union Conference on Freshwater Invasives- Networking for Strategy in 2013 is undertaken. The results indicate the need to strengthen parkcommunity relations,upgrade existing legislation,and boost the technical capacity of South African national parks to identify,detect,monitor and predict IAS invasions,both within the parks and their surroundings. This knowledge is important for developing future policies on IAS control in South Africa.
