Technology For Removal Of Antibiotic Resistance Genes In Sewage Based On Artificial Protein

Bacterial antibiotic resistance is a major threat to human health,and the spread of antibiotic resistance gene(Antimicrobial Resistance Gene,ARG)in water environment is one of the main ways of its transmission.Antibiotic-resistant bacteria(Antibiotics resistance bacteria,ARB)and antibiotic resistance genes have been widely detected in aqueous environment samples,and the transport of ARB or ARG between water environments may enhance antibiotic resistance in non-resistant bacterial communities through horizontal gene transfer process.Investigations were performed on previously known ARG removing techniques including conventional processing,advanced oxidation,electrochemical,and membrane bioreactor technology,and assessed their ARG removal efficiencies.Although some methods have a significant effect to eliminate the ARGs,these methods generally present serious limitations.With the widespread presence of antibiotic-resistant bacteria and antibiotic-resistant genes,it is urgent to find a better way to remove ARGsAfter analyzing previous methods to remove drug-resistant genes,this thesis proposed a new technology to remove drug-resistant genes in water environment by using DNA-binding proteins to specifically bind ARGs.Zinc finger structure is a DNA binding domain that can specifically bind to DNA,although the recognition specificity of a single zinc finger is very low,and it is easy to bind to other genes.However,by the use of six consecutive zinc fingers together,the DNA binding specificity is significantly improved.The designed structure was able to recognize a sequence of 18 bp of DNA,greatly reducing the off-target chance.Combining the zinc finger domain with other functional domains allows the creation of artificial zinc finger proteins that control and manipulate resistance genes.Artificial zinc finger transcription factors have previously been successfully used to control disease-related genes.Zinc finger nuclease domains(ZFNs)allow gene modification through sequence-specific double-strand breaks and endogenous repair processes.Therefore,we believe that the possibility of removing ARGs by DNA-binding proteins is very high.We first classified most antibiotic resistance genes in aquatic environments by phylogenetic tree analysis,and chose sulfonamides resistance sull gene and aminoglycaramines resistance genes aac(3)-Ib/aac(6’)-Ib ",aac(6’)-Ⅱd,aac(6’)-Ib-cr for attempts of artificial zinc finding containing protein design.The selected ARGs were analyzed by Zinc Finger design tool,and an 18 bp sequence was selected for the design of Zinc finger structure.After the design was completed,it was cloned into pET-21b,an expression vector with histidine label,and transformed into E.coli BL21(DE3)by chemical transformation.By optimizing the purification conditions of the protein,we obtained the DNA-binding protein with good purity and expression quantity.We then carried out EMSA and ITC experiments on the designed DNA-binding proteins and the corresponding ARGs in vitro,and obtained the binding affinity between DNA-binding protein and ARGs.Finally,the protein was cross-linked to the agarose gel by enzyme cross-linking,and the water containing ARG slowly flowed through to simulate the real environment,so as to obtain the binding efficiency of ARG.The full length of Sul binding protein contains 176 amino acids,with a size of about 19.8 kDa and an isoelectric point of 9.4,while the full length of Aac binding protein contains 176 amino acids,with a size of about 20.0 kDa and an isoelectric point of 9.58.EMSA assay showed that the binding efficiency of Sul binding protein to sull drug resistance gene was very good,with a KD value of 1.44 μM.while the binding efficiency of Aac binding protein was very poor.The curve obtained from ITC experiment of sull can be fitted,and the KD value is 9.06 μM.which is in the same order of magnitude as the KD value obtained from EMSA.After the protein is cross-linked to the agarose gel,the agarose gel is packed into a column,and the water containing the sull antibiotic resistance gene that flows before and after the collection is collected.The qPCR quantitative comparison shows that the binding efficiency is about 50%.Although the removal technology of antibiotic resistance genes has been studied for more than 50 years,there is still no efficient method developed for removal of ARG.Using DNA binding proteins specificity binding antibiotic resistance genes,we developed a method for specifically binding to antibiotic resistance genes,which can also remove ARGs by assembly with other functional domains,while maintaining an ecostability in aquatic environments.This method,however,is still challenged by limitations,such as the ability to only remove extracellular ARGs.This thesis is an attempt for ARGs removal in wastewater,in hopes for offering insights for the development of efficient antibiotic resistance gene removal in the future.