Preliminary Exploration Of Streptomyces Intelligent Transcription Regulation Network

Streptomyces produce diversity of natural products,which have a variety of biological activity and function,are treasury to find new drugs.The intelligent main source of equilibrium growth and production timely appropriate expression is delicate multi-level transcription regulatory networks(accurate DNA binding sites,the interaction between small molecules and transcription factors).To fully understand and know the mechanism of transcription regulation networks can help to obtain high-yield strains.Thus,we have taken advantages of ALPHA(amplified luminescent proximity homogeneous assays)technology,including high-throughput,homogeneous,high sensitivity and flexibility,low background,convenient miniaturization and automation to supplement the traditional methods(EMSA,DNaseI footprinting,surface plasmon resonance,chromatin immuno precipitation analysis,etc),and establish fast and high-throughput research methods for regulation networks.Avermectin,as a high-efficiency biological pesticide,derived from Streptomyces Avermitilis is an excellent representative of microbial medicine,and has great significance to ensure the safety of our food,agricultural products,animal husbandry and medical health.Furthermore,avermectin produced by China has protected 200 million Africans from river blindness.After penicillin and streptomycin,Satoshi omura from Japan,William C.Campbell from America,have gotten the favour of the 2015 Nobel Prize for the third time due to avermectin.1)In order to explore the new strategy for Streptomyces genome-wide transcription factor targets screening,we utilized the interaction of AvaRl and sav3705p in Streptomyces avermitilis which has already been confirmed,to establish a method platform to explore the interaction of transcription factors and DNA.This method can not only quickly detection the presence of interaction of transcription factors and DNA,but also can determine the precise binding sites of interaction via step by step truncated strategy,which represent the strength of the interaction.Furthermore,it can be applied to screen the interested target transcription factors among the whole genome,with the advantages of speediness and high-throughput.We utilized this method platform have found a new binding site,determined the precise binding sites rather than the protection sites,and the genome-wide targets of transcription factors AvaRl via the platform which can provided a valuable support to reveal the regulation network.2)A new method for the detection of chemicals has been developed based on the allosteric transcription factors(aTFs)which exist widely in prokaryotes and have the capability of sensing different chemicals.We first combined this characteristic of aTFs with ALPHA technology to develop a high-throughput detection platform for detection of new chemicals in vitro.This method utilizes micro porous plate to detect chemicals in liquid phase homogeneous system without the complicated cleaning steps,so that the high-throughput detection to chemicals has been realized stably.For the screening of terramycin high-producing strains,the most sensitive terramycin detection biosensor was obtained and the detection limit was as low as 0.03 nM.We also aimed at detecting uric acid which is the important clinical marker.Therefore,the most sensitive uric acid detection biosensor was built and detection limit was as low as 1 nM.Besides,this platform can also be used to develop new detection methods with high sensitivity for other chemicals,supporting the detection of clinical markers and development of relevant kit.