Part:BBa_K1828902:Experience

XrpA part

We measured the efficiency of biofilm production by S.mutans via overexpression of endogenous xrpA protein under pH - sensitive promoter. Nisin-Controlled Expression system was activated by addition of nisin polypeptide, and the promoter was induced by acetic acid-sodium acetate buffer (pH=5.5). The efficiency of construct was checked by measuring biofilm with spectrophotometer (Varioscan Flash Thermo Scientific).
There is a difference between S.mutans having XrpA construct and the one that lacks it. Data indicates that XrpA transformed S.mutans has low level of biofilm than wild type during 2, 6 and 8 hours time intervals.
Fig. 1 and Fig.2 show that the amount of biofilm formed by S.mutans (x, acid, n) was lower than in the rest of control samples.
It can be seen from all three figures that the addition of acetate buffer had inhibiting effect on the growth of bacteria. Therefore, the amount of biofilm formed by S.mutans (x, a) is lower than S.mutans (x, n), S.mutans (x) and S.mutans (wild). We hypothesized that the defense mechanisms that usually protect S.mutans from acid stress might not be activated at short time range. The measurements on Nunc Plates have certain limitations such as absence of coating for Gram + bacterial species, and loosing of not attached biofilm mass during simple staining. The more accurate results could be obtained from using electron scanning microscopy. However, the first two figures confirmed theoretical and practical results from studies performed by Kaspar et al. in 2015.

Figure 1. Measurements of absorbance of biofilm at 2 hours incubation.
x- S.mutans transformed with XrpA construct under pH- sensitive promoter.
nisin- S.mutans transformed with XrpA construct under pH-sensitive promoter + nisin polypeptide.
acid- S.mutans transformed with XrpA construct under pH-sensitive promoter + acetate buffet (the promoter is active).
wild- S.mutans without construct.

Figure 2. Measurements of absorbance of biofilm at 6 hours incubation.
Figure 3. Measurements of absorbance of biofilm at 8 hours incubation.

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