Design Of The Synthetic Self-Cleaving Aptazyme And Its Application On Gene Expression In Bacillus Subtilis

Bacillus subtilis,an important model organism of gram-positive bacteria,is widely used in industrial biotechnology and synthetic biology such as metabolic engineering,recombinant protein expression,and novel vaccine carriers.So B.subtilis was employeed as a promising chassis for synthetic biology in recent decades.Up to date,only few regulatory parts such as promoters and terminators were applied on gene expression in B.subtilis.Besides,the performance of conventional regulatory parts was easy to be influenced by genetic environment.Therefore,further application of B.subtilis in synthetic biology depends on the development of novel biological parts.RNA regulatory parts,which are independent on trans-acting factor,have great application potential due to their good orthogonality with hosts.This study successfully constructed aptazyme based on ribozyme and an artificial aptamer in B.subtilis and employeed this part to regulate gene expression.The main results are as follows:?1?A ribozyme-mediated gene expression plasmid pP43RZGFP was constructed on a vector containing the promoter P43 and a reporter gene GFP.And the influence of solvent DMSO and solute theophylline against ribozyme were tested.The results showed that strain PBP43RZGFP expressed GFP with a fluorescence intensity of 118 083±7 365 a.u.,while the inactivated ribozyme-containing strain PBP43inRZGFP had a fluorescence intensity of only22 048±109 a.u.,which indicates that RZ could regulate GFP expression in B.subtilis.After adding DMSO,the fluorescence intensity of PBP43RZGFP increased,while the fluorescence intensity of PBP43RZGFP decreased after theophylline addition.?2?Screening of communication module library which links the theophylline aptamer to ribozyme.33 of them were chosed for further study,and the WHBS parameters were used for correlation analysis.It was found that the leaky expression of GFP was significantly correlated with the WHBS parameter.The results indicated aptazyme can be rationally designed according to the WHBS parameters,and the sequence BG8?5'-GGT-32 nt-TCT-3'?was selected for subsequent research.The slected aptazyme was renamed as AZ.?3?Six different promoters P_rpoB,P_ylbp,P_spoVG,P_minC,P_sigW,and P_yqeZ,were combined with aptazyme and riboswitch to construct corresponding recombinant strains,and the regulatory properties were detected.The results showed that aptazyme?AZ?can all be activated.The combination of AZ and P_rpoB?rpoBAZ?had the highest induction rates,reaching 6.2.Similarly,the riboswitch E was matched with the same promoters,and the regulation function of riboswitch was analyzed.The promoter P_sigW and riboswitch E combination?sigWE?had the highest induction rate of GFP expression,reaching 16.8.Albeit induction ratio of riboswitch was higher than that of aptazyme,leaky expression of aptazyme was much lower,which showed aptazyme was more rigorous to gene expression.?4?In order to further expand the application of RNA regulatory elements,aptazymes and riboswitches were fused with different exogenous proteins mCherry,pullulanase and nattokinase.It was found that sigWE regulated mCherry with the highest induction rate?9.2?;P43E regulated pullulanase with the highest induction?32.8?,and the enzyme production level reached 81 U·mL^-1;P43AZ regulated nattokinase with the highest induction?2.9?.In this research,artificial aptazymes were successfully constructed.The properties of aptazymes were analyzed,and compared with that of riboswitch.The low level of leaky expression of aptazyme is important for designing complex genetic circuits in Bacillus subtilis.