Construction Of Random Mutant Library To Screen Sensitive Biological Elements For The Detection Of 2,4,6-Trinitrotoluene

Synthetic biology is a new interdisciplinary science in the 21st century,combining science and engineering to study the mechanism of life,which provides new technologies/ideas to solve the problems faced by human beings in the fields of medical treatment,environment and energy.At present,the construction of biosensor based on synthetic biology technology to monitor environmental pollutants has shown a good development trend.The core part of the biosensor is composed of a bio-recognition element and a report element.The bio-recognition element can induce signal expression of downstream reporter component after sensing and binding to the target compounds.What's more,the signal intensity indicates the induction intensity of bio-recognition element to inducer.Nowadays,the research of the report components is relatively mature,but the bio-recognition elements which can sense target compounds are still need to be dig,identified and optimized widely.We can say,the bio-recognition components are the basis of biosensors for selective determination.2,4,6-Trinitrotoluene(TNT)is a crystal nitrobenzene explosive material that is colorless,or pale yellow,and an important raw chemical material useful for the defense industry,mining,and infrastructure.At explosive manufacturing plants,battlefield sites and military training areas,TNT pollution has become a major environmental issue,which poses serious adverse effects on human health.Therefore,to develop an efficient,accurate detection of TNT tools are of significance on the minefield clearance and environmental monitoring.However,there are some limitations in the current chemical or physical detection methods,which almost all need expensive and complex equipments.As the biosensor has the advantages of low cost,high sensitivity,easy to carry and easy to operate,so it can be used as an auxiliary means of traditional methods to detect TNT.Under the guidance of synthetic biology,our main work in the study is to screen new bio-recognition elements which respond to TNT strongly,rapidly and specially.Our ultimate objective is to construct a whole biosensor by assembling the sensing elements we identified with reporter proteins,which can be served as monitoring tools for TNT-contaminated field.The major results of the paper can be divided into three parts:(1)Construction of screening platform with stringent repliconBased on the principle of differential fluorescence induction analysis,we should first construct a better platform to screen biological elements.We substitute the low-copy number replicon of pSC101 for the high-copy number replicon of pPROBE-TT.Then,the platform named 101-pPROBE,with stringent and broad host range replicon and a promoterless GFP were constructed.The bio-recognition elements can be inserted into the multiple cloning site and control the expression of GFP,and the fluorescence intensity represents the induction intensity of bio-recognition elements to target compounds.(2)Construct a random mutant promoter library to screen DNA partsBy analysing the commonness of promoter,we conserved the-35 region and-10 region,the rest was random synthesized completely.Then,a random mutant promoter library was constructed.After screening this library,new response element named 5M6A that exhibited strong induction response to TNT was identified.(3)Construct random mutant XylR library to screen protein partsThe XylR-Pu is a classic toluene catabolic pathway,which is from TOL plasmid of Pseudomonas putida.In the presence of toluene,the XylR regulatory protein can activate Pu promoter and thus induce expression of corresponding metabolic genes.The pathway was optimized and put into Escherichia coli to construct whole-cell biosensor to detect TNT.E.coli was chosen as chassis cell due to its genetic background was clear and it was simple to operate.pETDuet-1 was used as backbone to construct gene circuit of XylR-Pu,XylR was inserted in first multi cloning site.The second T7 promoter was substituted by Pu promoter and reporter gene of green fluorescent protein was under the control of Pu promoter.The fluorescent values can indicate the strength of the activation of XylR protein to Pu promoter.Then four series terminator was inserted between XylR and Pu to minimize background expression.Finally,the receptor domain of XylR protein was randomly mutated using sequential error prone PCR to construct a mutant library and to identify XylR mutants,which can be more sensitive and specific to TNT.After selection,one mutant protein named eX0-4 displayed better induction intensity,sensitivity and specificity to TNT.In summary,we obtained two new responsive elements that showed high sensitive and specific to TNT and that are promising as one of the core components of a biological system for building a new generation of biosensors.Meanwhile,the sensitivities of the two new sensing elements have all reached the leading level of similar components reported in the world.As such,these whole-cell/bacterial biosensors offer versatile and widely applicable methods for detecting the presence of a subtle level of toxin.The specificity or broad spectrum of this detection method provides the possibility to detect TNT and related compounds in real environmental samples,such as soils and plant tissues.