Biodesign Research to Advance the Principles and Applications of Biosystems Design

Over the course of civilization,humans have increasingly expanded their freedom to live a better life.In comparison with the primitive society,our modern society has many more choices of life-supporting resources,such as yearround food supply,permanent shelters,diverse energy sources,and effective preventive and curing medicine.However,our society is currently still heavily relying on the resources provided by Mother Nature,which cannot meet the future global needs in terms of both quantity and quality under the pressure of population growth,natural resource reduction,and environmental deterioration.For example,the food sources originating from plants,animals,or microbes do not have the nutrition balance for optimal human health[1–3].Climate change and environmental deterioration threaten the food security[4–6].Increasingly,infectious diseases(e.g.,HIV/AIDS),genetic diseases(e.g.,cancer),and improper lifestyle-related disorders(e.g.,obesity)become more prevalent and remain challenging to be prevented,controlled,and cured.Conventional medical technologies and modern medicine development are also meeting the ceiling.

Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli

Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy.Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane,soluble one-component repression systems provide several advantageous characteristics.Firstly,they are soluble and able to diffuse across the cytoplasm.Secondly,they are smaller and of lower complexity,enabling less taxing expression and optimization of fewer parts.Thirdly,repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors,potentially enabling implementation in different organisms.Herein,we present the design of the synthetic promoter P_(EL)that in combination with the light-regulated dimer EL222 constitutes a onecomponent repression system.Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter,we designed P_(EL)with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound.P_(EL)is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five.Further,alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible.The design of the P_(EL)EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems.Finally,we compare the P_(EL)-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.

Engineering a Circular Riboregulator in Escherichia coli

RNAs of different shapes and sizes,natural or synthetic,can regulate gene expression in prokaryotes and eukaryotes.Circular RNAs have recently appeared to be more widespread than previously thought,but their role in prokaryotes remains elusive.Here,by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme,we created a small noncoding RNA that self-splices to produce a circular riboregulator in Escherichia coli.We showed that the resulting riboregulator can trans-activate gene expression by interacting with a cis-repressed messenger RNA.We characterized the system with a fluorescent reporter and with an antibiotic resistance marker,and we modeled this novel posttranscriptional mechanism.This first reported example of a circular RNA regulating gene expression in E.coli adds to an increasing repertoire of RNA synthetic biology parts,and it highlights that topological molecules can play a role in the case of prokaryotic regulation.