Team:ECUST/Biofilm Remover

BIOFILM REMOVER

Description：

Dispersin B is a β-hexosaminidase that specifically hydrolyzes β-1,6-glycosidic linkages of acetylglucosamine polymers found in biofilm. This enzyme is derived from Aggregatibacter actinomycetemcomitans, which secretes adherent cells from mature biofilm colonies by secreting disperse B. The active site of Disperse B contains three highly conserved acidic residues: aspartic acid at residue 183 (D183), glutamic acid at residue 184 (E184), and glutamic acid at residue 332 (E332).

Blue color shows the E184；Pink color shows the D183；Yellow color shows the E332.

Design：

We hope that Dspb, like siderophore, is regulated by HSL, and when HSL is present, the dspb gene will express. It is also considered that the dspb enzyme activity is low, can be accumulated in the cells first, and then released by the subsequent lysis system.

Result：

In order to test the biofilm removal effect of DsPb, we constructed the vector pET28a-DsPb.

Figure 1. Construction of the expression vector pET28a-DSPB. The Kan represents kanamycin resistance marker. The part DSPB was inserted by the restriction site BamHI and XhoI

The plasmid was transformed into E. coli BL21, cultured at 37 °C for 12 h, and the plasmid was extracted and verified by PCR.

Figure 2. 1% Agarose Gel Electrophoresis of DNA extracted from the positive clones (a)and its identification by PCR(b), which shows that our vector was successfully constructed

To test the expression of DsPb, we cultured E. coli in LB medium containing 0.1% kan. E. coli was cultured at 37℃ and 220 rpm for all night, then inoculated into fresh medium and cultured until logarithmic phase.Add IPTG to final concentration of 20uM and culture E. coli over night at 25℃. Final OD600=0.437(diluted 16 fold)

Figure 3.The SDS of DSPB Lane 1,2 Lane 1,2 before induction Lane 3 cell cultue media Lane4,5,6 after induction

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