Part:BBa_K4817015:Experience
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Applications of BBa_K4817015
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The formation of microbial biofilm depends on the quorum sensing molecules AHLs, so the crystal violet staining results of the bacterial membrane treated with the protein solution can reflect the degradation effect of the protein on AHLs. The results of crystal violet staining show that AidH and AidH A147G/AidH A147V have good degradation effects on the biofilm of SRB. It shows that these proteins can quench quorum sensing molecules and thereby block biofilm formation.
For protein mutants, since the side chain of the 147th alanine residue of AidH may hinder the binding of the substrate to the active pocket, the degradation effect of AidH A147V with smaller steric hindrance is greater than that of AidH(WT). On the contrary, the degradation effect of AidH A147G, which is more sterically hindered, is worse than that of AidH(WT). The experimental results are consistent with the theoretical results.
Figure.5 Degradation effect of AidH crude extract at different concentrations on oxo-C6-HSL
4mL of AidH protein solution extracted from 30ml of bacterial solution, at dilution factors of 30ul/10ul/1ul (30ul in total), has a similar degradation effect on oxo-C6-HSL .
Figure.6 Degradation effect of AidH crude extract on different AHLs
Measure the AHL concentration with E.coli DH5α (with p15A-lux-sfGFP) , and reflect the AHL concentration by fluorescence intensity.
4mL of AidH/ AidH A147G/AidH A147V protein solution extracted from 30ml of bacterial solution, at dilution factors of 30ul/10ul/1ul (30ul in total), has a similar degradation effect on oxo-C6-HSL . For different AHLs, after adding 30ul of undiluted enzyme, the fluorescence intensity of all treated experimental groups was lower than that of the untreated experimental group. In general, the fluorescence intensity decreased overall.
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