Manipulation Of Intracellular C-di-GMP For Photoresponsive Systems And Quantitative Researches Of Bacterial Homologous Recombination

C-di-GMP is an important signal molecule in bacteria,which is mainly involved in regulation of bacterial adhesion behavior and formation of biofilm,especially in Pseudomonas aeruginosa.With the development of synthetic biology,especially photoresponsive system,more and more researches are involved in applications with c-di-GMP molecules.In the first part,based on c-di-GMP regulation,we designed two different light responsive system: single light responsive system and dual light responsive system.With the manipulation of single light responsive system,single cells were manipulated automaticly in real time.After further optimization of photoresponsive system,the dual light responsive system was established.Using static patterned illumination methods,reversible patterned bioprinting of bacterial biofilm was completed with living cells after further optimization in experimental system and experimental conditions.Due to the stronge modification potentials in engineered bacteria,the above two different bacteria stystems provided new methods and thoughs for the fields of novel anti-microbial surface and novel living bacteria based biomateirals.In the second part,a new system was designed to quantify intracellular homologous recombination rate based on a three-color fluorescent proteins system,which named Three-color Fluoresencent Proteins based Recombination Rate Calculator System,TFPRRCS.The accuracy of TFPRRC system was confirmed via PCR and further sequencing.Using the new methods from the established system,the intracellular homologous recombination rate was quantified quickly and accurately.Via the quantifications of homologous recombination rate to 16 recombination related mutants,it was found that the homologous recombination rate of recA mutant had decreased significantly in Pseudomonas aerugonisa,but the homologous recombination rate of uvrD and radA mutants had increased significantly in Pseudomonas aerugonisa,while the homologous recombination rate of uvrDrecA and radArecA mutants had come back to the low level in Pseudomonas aerugonisa.At the same time,the homologous recombination rate was quantified in different physiological conditions in Pseudomonas aerugonisa using the TFPRRC system.It was found that the homologous recombination rate of wild type had increased significantly in Pseudomonas aerugonisa under the certain concentration of ciprofloxacin,aslo the homologous recombination rate of mutant with high level of intercellular c-di-GMP had increased significantly in Pseudomonas aerugonisa.Meanwihle,to explore the effects on homologous recombination rate,homologous recombination rate was quantified the in different DNA structures using TFPRRC system,and it was found that the longer homologous sequences were,the more the homologous recombination rate had increased under the same length of sequences gaps,witch followed the exponential correlation in a certain duration,but the exponential correlation could disappear when the length of homologous sequences were longer or shorter than a certain value.We set up a mathematic model to fit perfectly the correlation between the length of homologous sequences and homologous recombination rate,which indicated that the energy resource in DNA homologous strands exchange came from the entropy change,and the strict matching of exchanged strands was needed.It was also found that there was no significant difference in homologous recombination rate in different homologous sequences length,different insertion positions of homologous sequences,different incubation time and different promoters and so on.