DNA Program The Assembly Of Bacteria And Regulating Their Quorum Sensing System

A long-standing goal in cell biology is to achieve precise and controllable manipulation of live cell populations.The ability to control the positioning,movement,and assembly of live cells with high spatial resolution in three-dimensional(3D)space is of great significance for studying cell-cell communication.Quorum sensing system plays an important role in the processes of microbial growth,division,reproduction,and resistance to external adverse environments.Therefore,the use of quorum sensing to regulate the biological phenotype of bacteria has been a hot spot in the field of microbiology.Bacterial quorum sensing can regulate the formation of bacterial biofilm,the enhancement of bioluminescence,the acquisition of antibiotic resistance,and the production of bacteriocin.Inspired by DNA nanotechnology,this thesis builds upon the principle of double-stranded DNA(ds DNA)base-pairing and utilizes the programmable nature of DNA structure to realize highly controlled assembly of E.coli and S.aureus enable us to regulate the formation of a biofilm,one of the phenotypes of the bacterial population.In this thesis,we first modified bacterial surface with functional DNA using metabolic labeling and click chemistry.The target bacteria used added azido sugar as one of the polysaccharide components to synthesize cell wall with azide modification.In the next step,single-stranded DNA(ss DNA)with dibenzocyclooctyne(DBCO)terminal modification is coupled the azide groups on the surface of bacteria through copper-free cycloaddition reaction,forming a layer of DNA strands on bacteria surface via covalent bonds.Under appropriate conditions,DNA hybridizations at the interface of neighboring bacteria regulates the cell organization to achieve controlled assembly of target bacteria.At the same time,bacterial cells remain biological activity during the assembly process.Since the concentration of signal molecules secreted increases at higher cell density,when the concentration of the signal molecule reaches threshold,it can stimulate the bacterial cell to initiate a series of related gene expressions,thereby changing the biological characteristics,such as forming a biofilm and generating bioluminescence.In DNA-mediated bacteria assemblies,ds DNA linkage shortens the distance between neighboring bacteria and increases the local concentration of signaling molecules,thereby promoting the formation of bacterial biofilms.The methods and key results of this thesis are stated as follows:(1)We used metabolic labeling approach to achieve DNA modifications on the surface of bacteria.During the metabolic labeling process,experimental conditions such as sugar type,concentration,and incubation time of the added azido-sugars have significant effects on the azide surface modification efficiency.We used flow cytometry,laser confocal microscopy,and other characterization methods to measure azide labeling efficiency on bacterial surface under different conditions.The results showed that reaction in culture medium containing 1 m M Glu-N₃ and shaking at 37?for 20hours generated high labeling efficiency on E.coli.Under the laser confocal microscope,a number of intact cells with red fluorescence from DBCO-Cy3 dye were visible to the naked eye.By quantifying the number of DBCO-Cy3 fluorescent dyes modified on the surface of bacteria,we are able to calculate the average number of azide groups on one bacterial cell after metabolic labeling,thereby estimating the theoretical amount of DNA strands required for surface modification.We further studied the DNA concentrations effects on the modification efficiency.In order to demonstrate whether the bacterial surface was successfully coupled with target DNA strands,we designed two control groups containing unmodified and non-complementary bacteria to study the bacterial surface DNA modification using flow cytometry,laser confocal microscopy,and other characterization methods.The results showed that DNA strands were successfully connected to the bacterial surface through click chemistry,maintaining the sequence recognition ability to pair with complementary strands.(2)We successfully demonstrated the DNA-mediated programmable assembly of E.coli,and systematically studied the effects of DNA sequence,salt ion concentration,DNA concentration,and other experimental conditions on the formation of bacterial assemblies.We used Coulter counters,dynamic light scattering,and laser confocal microscopy to characterize the size and morphology of formed bacterial assemblies.The results showed that the size of obtained E.coli assemblies ranging between ca.2-30(?)m with a maximum size of ca.40(?)m.In order to study the biological activity and quorum sensing effects of the assembled bacteria,we studied the biofilm formation of the E.coli assemblies.We quantified the amount of biofilms produced by two complementary groups and one non-complementary group using crystal violet staining.The results showed that,at low initial bacteria concentration,the complementary group produced more biofilm than the non-complementary group,and different sequence designs in the complementary group also showed different effects in tuning the biofilm formation.(3)To demonstrate the generality of the DNA-mediated bacteria assembly,we applied the strategy to a class of iconic Gram-positive bacteria,S.aureus,successfully forming micrometer scale S.aureus assemblies.We used the same protocols to metabolically label S.aureus with azide groups,modify target DNA strands on the bacteria surface,and further program the assembly of S.aureus through the DNA hybridizations.By using Coulter counters,dynamic light scattering,flow cytometry and laser confocal microscopy,we systematically characterized the size and morphology of the S.aureus assemblies.The results showed that DNA-labelled S.aureus were successfully assembled into micrometer scale clusters.The size of the assemblies ranged from ca.10-30(?)m with the largest cluster reaching ca.45(?)m.Compared with Gram-negative E.coli,Gram-positive S.aureus showed higher assembly efficiency and larger average cluster sizes.In summary,in this thesis we reported the DNA-mediated programmable assembly of live E.coli and S.aureus cells based on the basic principles of DNA nanotechnology.Live bacterial assemblies with size distribution of 20-40(?)m were obtained.The bacteria in the assembled clusters are still alive and maintain biological activity.By quantifying the biofilm produced by the E.coli assemblies,we found that E.coli assembly enhanced the biofilm formation process compared to the non-complementary group at low initial bacteria concentration.The DNA-mediated assembly of bacteria strategy developed in this thesis provides a powerful tool for future study of bacterial quorum sensing.