Research Of The Quorum Sensing Mechanism Of Pseudomonas Aeruginosa And Multicolour Imaging

Pseudomonas aeruginosa is an opportunistic pathogens,widely distributed in na-ture.It contains three types of quorum sensing systems mainly including the las,rhl and PQS.They regulate many downstream genes such as bacterial toxins,elastase,biolu-minescence and etc.So the quorum sensing(QS)gene pathway is one of the important targets for drug design.Rhamnolipid,the production of rhl system,is a kind of biosur-factant,which assists bacteria process swarming motility.Due to its amphiphilic prop-erty,past research considered it may help hydrophobic signal molecules diffuse between bacteria.In order to test the presence of C4-HSL and C12-HSL,which are quorum sens-ing signaling molecules,we introduced the Prhll promoter because it could respond to both signals.Prhl belongs to the weak promoter,and the second chapter put forward a new type of double fluorescent colour gene reporter,which made double amplification both in the forward and reverse directions.It effectively improved the signal-to-noise ratio.By adding exogenous C4-HSL to induce JP2-HJD15B-PrhlI-Tn7 strains,the un-even inducing phenomenon of Prhll was detected.We speculated that something of extracellular secretes blocked signaling molecules from passing in and out of the bac-teria.After screening,rhlA knocked out or overexpressed had significantly effect on absorbing the signal molecules.To a certain extent,rhamnolipid can preclude signal molecules from difusing in and out of the bacteria.Signals are not evenly distributed all over the biofilm,but dispersed in the formation of rhamnolipid compartment,thus to make local concentration unequal.It made the bacterium generate signal molecules only for itself and it may be a new mechanism for bacteria to inhibit social cheaters appearance.Up to date,there are many fluorescent proteins with different light spectra are avail-able for the microscopy research,but multicolor imaging strategies are still mainly fo-cusing on using these fluorescent proteins with little light spectrum interference.Here,a new fluorescence correction method was reported in the chapter three and four which could successfully separate up to five color fluorescent proteins mixture.The new flu-orescence correction was made according to the equation given in this paper that every single fluorescent proteins could be tested its absolute concentration after correction.Based on the previous experiment,different fluorescent proteins were purified and the standard curve was made by changing the excitation wavelength,exposure time,flu-orescent channels.So the fluorescent correction equation could be solved and the ab-solute fluorescent concentration was got.Also a fast gene reporter assembly kit was developed in this thesis and it became very useful and meaningful:different fluores-cent protein gene sequences with ribosome binding sites and terminators were designed as one component,so it was easy to assemble gene reporters with different color as the experiment required.By using the kit and correction method,quorum sensing reporters of pseudomonas aeruginosa with four colors(sfGFP,mScarletl,Cyofp and Venus)and another one color(sm URFP)acting as the calculating mask were constructed and tested.Multicolor imaging strategy of this paper can provide a new idea to solve more biolog-ical problems.In summary,this study closely focused on the Pseudomonas aeruginosa quorum-sensing aspects,and there were technological improvements on gene reporter construc-tions and multicolor imaging.By using these methods,we found rhamnolipids could preclude QS signal molecule functions.The research and technology above laid a solid foundation for future exploration.