Proliferation Of Bacteria In The Confined Space Of Giant Unilamellar Vesicles

Traditional bacterial culture monitoring is carried out in an open system,which is hard to control the culture conditions and analyze a single or a small number of bacteria.With the advancement of culture technology,more attention has been paid to bacterial culture at the single-cell level to intuitively analyze bacterial behavior.Giant unilamellar vesicles are formed by self-assembly of phospholipid molecules,which can provide a controllable closed micro-compartment with limited space.The exchange of internal and external substances can be achieved by modifying the phospholipid double-layer membranes,which can be used as a micro-culture chamber for bacteria culture.The size and internal space of giant unilamellar vesicles are limited,so the growth of bacteria would be different from that in open space.The vesicles can help the controllable reaction via exchanging substances with the outside world.In this thesis,giant phospholipid vesicles were used to encapsulate bacteria and to investigate the protein expression in E.coli when external signal molecules going into the vesicles.And the proliferation of E.coli and B.subtilis inside the vesicles was observed and simulated using mathematical models by comparing those in the open space and the confi ned space.Firstly,the giant unilamellar vesicles were prepared by electroforming method and emulsion transfer method to encapsulate nutrients and E.coli.Fluorescence microscopy results showed that the bacteria-containing vesicles prepared by the electroforming method suffered from external impurities and great influence by the salt ions.While there were only a small amount of bacteria in the external solution of the vesicles obtained by the emulsion transfer method,which is hardly affected by salt ions.So the emulsion transfer method is chosen for encapsulation.Subsequently,the preparation conditions were optimized,including the vortex time and centrifugal speed on encapsulation,the phospholipid composition and substrate modification on the stability of vesicles,and the relationship between the concentration of bacterial solution and the number of encapsulated bacteria.The results showed that with the increasing vortex time,the average size of vesicles became smaller and the distribution was uniform;with the increasing centrifugal force,it was easier to form phospholipid dots and phospholipid clusters;cholesterol-modified POPC and glass slide substrate modified by phospholipid membrane or bovine serum albumin are beneficial to improving the s tability of vesicles.The greater the concentration of the bacterial solution is,the greater the number of bacteria encapsulated in the vesicle is.The exchange characterization of encapsulated bacterial vesicles were studied.The melittin is added to induce the protein channel on phospholipid membrane,and then the IPTG inducer was added into the outer solution to diffuse into the vesicle and induce E.coli to express green fluorescent protein,confirming the controllable substrate exchange inside and outside of the vesicles.Based on the study,a four-parameter log-Logistic mathematical model was used to fit the growth curve of bacteria in open space and confined space.Through analysis,it is found that the adjustment period of E.coli in the confined space of vesicles is shorter than that in the open space,and the growth rate of E.coli is slower than that in the open space.Compared with the open space,the bacteria,multiplied in the confined space,are shorter and less likely to form filaments.