Background:Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms,like human diseases and microbial productivities of target bioproducts.Methods:Here,we develop...Background:Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms,like human diseases and microbial productivities of target bioproducts.Methods:Here,we developed a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualised the evolutionary trajectories of gene repeats.We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification modules.Results:This system delivered multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection.Inducing the toxin(RelE)expressing via a copper(II)-inducible CUP1 promoter successfully drove the in-situ gene amplification of the antitoxin(RelB)module,resulting in~40 copies of a green fluorescence reporter gene per copy of genome.Copy-number changes,copy-number increase and copy-number decrease,and stable maintenance were visualised using the green fluorescence protein and blue chromoprotein AeBlue as reporters.Copy-number increases happened spontaneously and independent on a selection pressure.Increased copy number was quickly enriched through toxin-antitoxin-mediated selection.Conclusion:In summary,the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications.展开更多
为实现鳗败血假单胞菌(Pseudomonas anguilliseptica,PA)早期感染的快速诊断,基于recA基因建立了2种检测方法:SYBR Green I实时荧光定量PCR(SYBR Green I real-time quantitative PCR)和重组酶介导等温扩增结合侧流层析试纸条(Recombina...为实现鳗败血假单胞菌(Pseudomonas anguilliseptica,PA)早期感染的快速诊断,基于recA基因建立了2种检测方法:SYBR Green I实时荧光定量PCR(SYBR Green I real-time quantitative PCR)和重组酶介导等温扩增结合侧流层析试纸条(Recombinase-mediated isothermal amplification combined with lateral flow dipstick,RAA-LFD)。以PA的管家基因recA为靶标,设计筛选出1对qPCR特异性引物、1对RAA特异性引物和RAA探针,并通过同源重组构建标准品质粒pUC18-recA,以建立2种检测方法。将所建立的方法应用于PA感染的大口黑鲈(Micropterus salmoides)组织样本检测,并测定PA载量。结果表明,建立的qPCR方法最低DNA检测浓度为2.816×10^(2)拷贝·μL^(-1),模板量与Ct值在构建的标准曲线中呈现良好的线性关系(r^(2)=0.9992),且具有较强的特异性和较高的稳定性;RAA-LFD方法的最低DNA检测浓度为2.816×10^(4)拷贝·μL^(-1),检测时间最快可达15 min,显色较为稳定且特异性强。应用结果显示,qPCR和RAALFD方法的阳性样本检出率分别为87.50%和85.00%,较普通PCR方法明显提高;其中,qPCR方法可准确测定PA感染宿主组织中的菌体载量,肾中的载量最高,达3.533×10^(7)拷贝·ng^(-1)。建立的2种方法特异性均较好,其中qPCR方法灵敏性更高,RAA-LFD方法则时效性更强,均可用于PA早期感染的检测,且qPCR方法还可对感染宿主体内的菌体载量进行定量分析。展开更多
With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a c...With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.展开更多
基金supported partially by the Australian Government through the Australian Research Council Centres of Excellence funding scheme(project CE200100029)。
文摘Background:Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms,like human diseases and microbial productivities of target bioproducts.Methods:Here,we developed a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualised the evolutionary trajectories of gene repeats.We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification modules.Results:This system delivered multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection.Inducing the toxin(RelE)expressing via a copper(II)-inducible CUP1 promoter successfully drove the in-situ gene amplification of the antitoxin(RelB)module,resulting in~40 copies of a green fluorescence reporter gene per copy of genome.Copy-number changes,copy-number increase and copy-number decrease,and stable maintenance were visualised using the green fluorescence protein and blue chromoprotein AeBlue as reporters.Copy-number increases happened spontaneously and independent on a selection pressure.Increased copy number was quickly enriched through toxin-antitoxin-mediated selection.Conclusion:In summary,the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications.
文摘为实现鳗败血假单胞菌(Pseudomonas anguilliseptica,PA)早期感染的快速诊断,基于recA基因建立了2种检测方法:SYBR Green I实时荧光定量PCR(SYBR Green I real-time quantitative PCR)和重组酶介导等温扩增结合侧流层析试纸条(Recombinase-mediated isothermal amplification combined with lateral flow dipstick,RAA-LFD)。以PA的管家基因recA为靶标,设计筛选出1对qPCR特异性引物、1对RAA特异性引物和RAA探针,并通过同源重组构建标准品质粒pUC18-recA,以建立2种检测方法。将所建立的方法应用于PA感染的大口黑鲈(Micropterus salmoides)组织样本检测,并测定PA载量。结果表明,建立的qPCR方法最低DNA检测浓度为2.816×10^(2)拷贝·μL^(-1),模板量与Ct值在构建的标准曲线中呈现良好的线性关系(r^(2)=0.9992),且具有较强的特异性和较高的稳定性;RAA-LFD方法的最低DNA检测浓度为2.816×10^(4)拷贝·μL^(-1),检测时间最快可达15 min,显色较为稳定且特异性强。应用结果显示,qPCR和RAALFD方法的阳性样本检出率分别为87.50%和85.00%,较普通PCR方法明显提高;其中,qPCR方法可准确测定PA感染宿主组织中的菌体载量,肾中的载量最高,达3.533×10^(7)拷贝·ng^(-1)。建立的2种方法特异性均较好,其中qPCR方法灵敏性更高,RAA-LFD方法则时效性更强,均可用于PA早期感染的检测,且qPCR方法还可对感染宿主体内的菌体载量进行定量分析。
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195)。
文摘With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.
