Team:NEFU China
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AI-2 Quantification
E.coli and Bacillus sp. are the main pathogens in spoiled yogurt. Detection of these pathogens can be used to identify whether yogurt has got bacterial contamination. The traditional detecting methods contain some cumbersome steps, need some expensive reagents and still have high inaccuracy. We checking the references, we found that autoinducer 2 (AI-2), a signal molecule in the quorum sensing system in bacteria, can also be used as a signal detected by our engineered Lactobacillus. Thus, we wanted to measure AI-2 produced by pathogens in spoiled yogurt and then analyzed the relationship between AI-2 output and bacterial growth.
The molecular structure of AI-2 is somewhat similar to ascorbic acid. It was expected that AI-2 would also act as a reducing agent and reduce Fe(III) ions in the presence of 1,10-phenanthroline to form the orange-red colored [(o-phen)3 Fe(II)]SO4 ferroin complex that could be quantified colorimetrically.
Protocol:
1 Inoculating
E.coli and Bacillus were inoculated into LB media (2%, v/v), respectively, and shaken overnight at 37°C.
2 Sampling
After incubation, the media were centrifuged at 6000 g for 3 min. the supernatant was collected and was filtered through a 0.22μm membrane
3 AI-2 Quantification
(1) Preparing the working solution: A working solution of 10 mM 1, 10-phenanthroline/3.32 mM Fe (III) was prepared by dissolving 0.198 g of 1,10-phenanthroline in 50 ml of deionized distilled water. The solution was adjusted to pH 2 using 1M HCl. Ferric ammonium sulphate (0.16g) was added and the solution was brought to 100 ml using deionized distilled water.
(2) For the Fe (III) ion reduction test, 1ml of the cell free supernatant was mixed with 1 ml to develop the full color. The solution was then diluted to 5ml and was filtered through a 0.22 μm membrane，and scanned for the absorption spectrum against a blank solution within 3 min using a Lambda 25 UV/VIS spectrometer.
Results
E.coli and Bacillus sp. are the representative strains of Gram-negative and Gram-positive bacteria. We verified that both of them can produce AI-2 during their growth process. Additionally, AI-2 production by both of the two strains is significantly positive correlated with bacterial growth in the first two hours. All the results showed that AI-2 could be produced by both Gram-negative and Gram-positive strains. Thus, our bio-detecting system might not only be used in Gram-negative bacteria detection, but also Gram-positive bacteria detection.