Construction And Efficacy Evaluation Of Boolean Logic Gates Based On Dual-vector Circuits

Objective:In order to lay the foundation for the precise regulation of gene circuits,the regulation effects of different activated and inhibitory riboswitches were established and compared;The Boolean logic gate model is designed by using the multivariate gene regulatory element,and the complex logic operation is introduced to realize the precise regulation of gene expression,and then to optimize the specific metabolic pathway.Methods:1.The green fluorescent protein amcyan expression vector modulated was constructed by different riboswitches（addA and M6,TPP and btuB）,the vectors were transferred into the MG1655 strain and the culture time of the strain was determined.The expression level of amcyan under different ligand concentration was analyzed by fluorescence microscope,RT-qPCR and relative fluorescence intensity,then compared the expression level of vector without riboswitch.The appropriate IPTG concentration was selected to regulate subsequent experiments;Under the above induction of IPTG concentration,ligands corresponding to different riboswitches were added,and the expression of green fluorescent protein amcyan was regulated at different concentrations of ligands.Then the optimal ligand inducing concentration for each riboswitch was selected to analyze the dynamic control performance.2.We organized a genetic circuit logic gate model by using lac promoter,riboswitch（M6,addA,TPP,btuB,LysC,c-di-GMP）,phage tR1 transcription terminator,Pbe promoter and controlled genes(pleD,lysC^for)in a dual vector circuit.Finally,we constucted four logic gates,including“AND”,“NAND”,“OR”and“NOR”,the expected efficacy of which were verified by fluorescence microscope,relative fluorescence intensity,RT-qPCR,HPLC,and LC-MS/MS.Results:1.Under the induction of IPTG,the expression of green fluorescent protein increased with the increase of IPTG concentration,and the green fluorescence expression was highest when IPTG concentration was 1 mM.When adding 1mM IPTG concentration,under the control of addA and M6activated riboswitches,the expression of green fluorescent protein rised with the increase of ligand concentration,and addA riboswitch had more dynamic regulatory effect than M6 riboswitch.On the contrary,under the control of TPP and btuB inhibitory riboswitches,the expression of green fluorescence decreased with the increase of ligand concentration,and the dynamic regulation of btuB riboswitch was slightly greater than that of TPP riboswitch.2.The four kinds of logic gates were successfully built in E.coli using a logic gate model.In the“AND”logic gate operation,the simultaneous application of TPP and AdoCbl led to a maximum fluorescence intensity.The other application of TPP and AdoCbl promoted lysC^for gene expression and increased the content of L-lysine,which then turned“OFF”the lysC riboswitch and inhibited the expression of the reporter gene.In the“NAND”logic gate operation,the simultaneous application of TPP and AdoCbl resulted in a minimum fluorescence intensity.The other application of TPP and AdoCbl induced the expression of pleD and increased the content of c-di-GMP,which turned“ON”the c-di-GMP riboswitch and increased the expression of the reporter gene.In the“OR”logic gate operation,the fluorescence intensity was lowest without Amm and 2-AP.The other application of Amm and 2-AP led to the expression of pleD and increased the content of c-di-GMP,which turned“ON”the c-di-GMP riboswitch and exposed RBS,so as to increase the expression of the reporter gene.In the“NOR”logic gate operation,the fluorescence intensity was maximum without Amm and 2-AP.The other application of Amm and 2-AP led to the expression of lysC^for and increased the content of L-lysine,which then turned“OFF”the lysC riboswitch and inhibited the expression of the reporter gene.Conclusion:1.The regulation efficacy of different riboswitches which have the same mechanism is different.Activated riboswitch addA and inhibitory riboswitch btuB with dynamic regulation and control advantages are more suitable for precise metabolism regulation and target gene expression in Escherichia coli.2.The designed logic gate model was successfully used for the construction of gene circuit logic gates in E.coli.The Conversion of regulatory elements with different mechanisms formed a genetic circuit containing different logical relationships,and each logic gate circuit（“AND”,“NAND”,“OR”,“NOR”）achieved the expected operational efficiency and regulatory characteristics.Our study provided novel insights into designing gene circuits and controllingthe metabolic pathways precisely,which showed great potential in biological computation and synthetic biology.