Construction Of Luminescent E. Coli With Lux Gene And Study Of Its Biofilm

The luciferase was encoded and synthesized by Lux luminescent genes. The substrate will react with the luciferase and produced visible blue-green light at the wavelength of450-490 nm. As a reporter gene, it has been used in many fields, such as online observation,environmental monitoring, food safety control, and in vivo animal testing. Bacterial biofilm is a kind of self-protective state formed by the stress reaction of the bacteria to the harsh environment, which is difficult to remove and kill after biofilm formation. This brings harm to the food processing, medical treatment and other fields. With a good ability of produce biofilm, E. coli DH5α which is introduced the plasmid pHSG396-P.luxCDABE(carrying lux gene of Photorhabdus luminescens) was selected to finish the research. By the method of determining luminescent, selecting blue-white, restriction endonuclease digestion and AGE(agarose gel electrophoresis) of verification, the recombinant luminescent E. coli was screened to study biofilms. It was successfully constructed a good ability to form biofilms of recombinant luminescent E. coli model.The physiological characteristics and the relationship between the biological membrane and the luminescent intensity of recombinant luminescent E. coli were studied at a deeper level. The conclusions were as follows:1. The physiological characteristics of recombinant luminescent E. coli(1) The growth curve and luminescence curve of recombinant luminescent E. coli were cultured with fresh LB liquid culture medium. The results showed that the adjustment phrase and logarithmic phrase of recombinant luminescent E. coli was 0-3rd h and 4-8th h respectively. The recombinant bacteria started its stationary phrase at 9th h. During the adjustment phrase and initial logarithmic phrase, the light emitted by the bacteria was weak.But it increased sharply when the recombinant bacteria grew into the logarithmic phase, and the luminescent reached the maximum value 2510 RLU at 9th h. After that, the luminescent intensity of the recombinant bacteria faded away gradually.(2) The OD600 nm value and luminous intensity of recombinant luminescent E. coli was determined under different cultureconditions(IPTG concentration, pH, Cml concentration, decanal concentration, temperature).The results showed that IPTG has a smaller role in promoting luminescence when the amount is less than 0.2 mg/mL, while has an inhibition effect when more than this amount. The recombinant luminescent E. coli has capable of emitting light when the pH of culture medium between 5 to 9, OD600 nm and luminous intensity of it both reached the maximum value when the pH was 7. Under the same growth concentration, the luminous intensity of the weak alkaline environment(pH=8) was higher than that of the weak acid environment(pH=6). Cml has a slight impact on the growth and the luminous intensity of recombinant bacteria when the amount of it was 0-37.5 μg/mL. Beyond this range, it has a certain inhibitory effect on its growth. Adding decanal has no positive effect on recombinant bacteria luminescence. The highest tolerance temperature of the recombinant bacteria was 43, and the OD600 nm value and luminous intensity appeared maximum value. The luminescence intensity of the recombinant bacteria was determined at room temperature. The results indicated that the luminescence intensity has a decreased trend in the beginning 15 min, and then leveled off. The luminescence intensity was always above 400 RLU within 2 hours.2. The relationship between the biofilm and the luminescent intensity of recombinant luminescent E. coli(1) In this study, the production of recombinant luminescent E. coli biofilm under four factors(pH, D-mannose, bacterial suspension concentration, Cml concentration) were explored. The methods of luminescent and crystal violet staining were adopted to determine the production of biofilm. The results revealed that the weakly alkaline conditions(pH=8)was more conducive to the growth of biofilm than the neutral conditions(pH=7). D-mannose was necessary for the cultivation of recombinant bacteria biofilm, and the amount of biofilm formation was the largest when the addition of D-mannose was 2.0 %. The OD600 nm value of the bacterial suspension was 0.3, which was the most favorable for the formation of biofilm.Too high and too low was not conducive to the growth of biofilm. The optimum Cml concentration of recombinant luminescent E. coli to produce biofilm was 12.5 μg/mL in this experiment.(2) The data of luminescent and crystal violet staining analyzed by regression analysis. The results showed that their data had good linear correlation(R-Sq > 90%). Under appropriate culture conditions, luminescent labeling can also measure the formation of bacterial biofilms to a certain extent.