The Development Of RNA Replication System And Its Correlation With Host Cell

Metabolic engineering is a booming area which people can used artificial designed and modified microorganisms to produce products.However the development of techniques to manupitate microorganism metabolic pathway is the first step of this area.To most circumstances in microorganism modification,how to express an exogenous artificial gene efficiently in target strain is the most important challenge we faced.Here our project develops a new method to enhance target gene expression in bacteria.Previous methods to enhance protein expression mainly focus on the increasing of gene copy number,transcription efficiency,mRNA stability or transcription efficiency.However in our project,we first propose that we can increasing target gene expression by directly amplified the mRNA of target gene using RNA replication procedure.The advantage of our method is that RNA replication increase mRNA level directly,with no conflict to any other method.In another word,you can also regulate the promoter or RBS in the replicative mRNA to get a higher expression in total.In our project,we first construct a replicative sequence according to Q? phage sequence.Then we optimize the replicative sequence to form a RNA replication system to enhance target gene.We also confirm the replicability of replication system and its effectiveness and universality to enhance target gene.Besides,we also analyze the cross talk between our RNA replication system and host bacteria.We find that the host effect interfere the efficiency of RNA replication system significantly,and this inhibition can be solved by knock out the host Rnc gene.By the way,we also use two target metabolite except reporter gene,ALA and PHB to test our system.We also show increasement of production of ALA or PHB using our system.Besides in E.coli,we also confirm that our system can also work in another gram positive strain Lb.caseil Zhang.In all,in our project we demonstrate a new method to increase gene expression by RNA replication system,and our attempt indicated that our method have the potential to be a universal method in metabolic engineering area.