Riboswitch CDNA In Vitro Assembly And Database Construction

Riboswitches are called nucleic switches, which are widely founded in various organisms. They can be considered as a class of structure of RNA sensitive to small metabolites and can regulate genes expression at the level of transcription and translation by binding with the small metabolites. Usually they locate at the 5'end of untranscription regions of mRNA (5'- untranslated regions, UTR) that do not code for proteins and can directly bind small metabolites without any protein factors, subsequently, they affect the activities of mRNA and regulate protein related small metabolites'expression by conformations rearrangement. Generally the small metabolites specifically binding with them are lysine, flavin mononucleotides (FMN), thiamine pyrophosphate (TPP), glycine, Cobalamin and so on. The area of small molecules binding with is called as aptamer domain (AD).The development of synthetic biology makes synthetic genes possible. It is time-consuming and money-spending using the traditional method of ordering base sequences to synthesize prospect genes or genome, besides the traditional method has high error frequency and it is need amount of short oligonucleotides for parallel gene construction. But with the application of microfluidic PicoArray technology it becomes relatively simple and effective for parallel oligos syntheses. So it simplifies the process of gene synthesis providing fragments for oligos assembling.We introduced the complete process from oligos to full-length Riboswitch gene. Firstly, took the controllable microfluidic PicoArray parallel synthesis Riboswitch oligos (these oligos have already been artificially designed and modified) as material and then recycled them after amplification and purification, mixed the oligos and digested with enzymes, and then ligated and assembled them after purification, lastly, amplified them by poly chain reaction and detected by electrophoresis. We testified the assembling result in vitra by ligation of targeted genes with vector, transformation, and clone and plasmid extraction and obtained the related data through the process of plasmid extraction, poly chain reaction, target genes'recovery and detection, quantity's measurement. Predict the secondary structure of RNA sequences corresponding to DNA by M-fold method, and then find the secondary folding structure of the conservative regions and domains of Riboswitch binding with ligands. Lastly, construct corresponding Riboswitch library according to different ligands and combination with the prediction of the relative data, the secondary folding structure of Riboswitch'DNA and analysis of aptamer conservative regions, all of this can lay basis for subsequent study on Riboswitch'structure and function.