Difference between revisions of "Team:ECUST/Biofilm Remover"

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<p>The expression of DsPb was verified by SDS-PAGE(Figure 3). The cell culture medium was blank control, and the pre-induction cell supernatant was used as a negative control. </p>
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<p>In the figure 3 we can see that there are bands at about 37 kDa, these band of  experimental groups are thicker than the negative control. And our target protein is about 40kDa. We think the protein here may be DsPb. </p>
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<p>Then, we tested whether the engineered bacteria expressed active DsPb through enzyme activity experiments. </p>
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<p>4-nitrophenyl-N-acetyl -β-D-glucosaminide(NP-GlcNAc) is hydrolysed by DspB and become 4-nitrophenol with the maximum light absorption at 405 nm. 20 mLbacteria solution is disrupted and acted supernatant as DsPb enzyme solution.
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We washed and shred the bacterial solution after induction, and took the supernatant as the enzyme reaction solution of DsPb while NP-GlcNAc working as a substrate.The enzyme activity experiment was carried out by using 100 μL of enzyme reaction solution and 100 μL of substrate solution (substrate concentration is 5 mM). </p>
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<img src=https://static.igem.org/mediawiki/2018/b/b8/T--ECUST--result--biofilm_remove_BIOFILM4.jpg" alt=" class="B4">
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<figcaption><b>Figure 4：DSPB enzyme activity assay curve
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<p>The enzyme activity of the supernatant calculated by the enzyme activity calculation formulais (Activity/(U/mL)=(kOD405-kseldecomposition)*18231.26 *dilution ratio) is 66.363U/mL. </p>
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<p>The experiment proved that the recombinant bacteria expressed active DSPB, and we used the E. coli supernatant with DSPB activity to carry out the biofilm removal experiment.DH5a was cultured overnight in LB at 37 ° C and 220 rpm, and transferred to a 96-well plate at 37 ° C and cultured for 48 h, then discarded the supernatant .The biofilm was washed with PBS, and 200 uL of the reaction solution was added to react for the whole night. Add crystal violet to stain, wash the solution into the new well plate after alcohol washing, and measure the absorbance at 570 nm. The smaller the absorbance value, the better the membrane removal effect. </p>
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<img src="https://static.igem.org/mediawiki/2018/2/2c/T--ECUST--result--biofilm_remove_BIOFILM5.jpg" alt=" class="B4">
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<figcaption><b>Figure 4.(a) Crystal violet staining results of different reaction solutions added to biofilm
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          (b) Biofilm removal rate of different reaction solutions added to biofilm
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          (c) DSPB biofilm-removing curve
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OD570=51.96-51.24t0.0051
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R2=0.98385
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<p>From Fig.5, The supernatant of the recombinant E. coli  has the highest biofilm removal rate, and the biofilm removal effect is getting better with time. </p>
 
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Revision as of 20:15, 17 October 2018

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).

Figure1.1 OD600 reference point tab
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.

Figure1.1 OD600 reference point tab

Result：

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

class=
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.

class=
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)

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

The expression of DsPb was verified by SDS-PAGE(Figure 3). The cell culture medium was blank control, and the pre-induction cell supernatant was used as a negative control.

In the figure 3 we can see that there are bands at about 37 kDa, these band of experimental groups are thicker than the negative control. And our target protein is about 40kDa. We think the protein here may be DsPb.

Then, we tested whether the engineered bacteria expressed active DsPb through enzyme activity experiments.

4-nitrophenyl-N-acetyl -β-D-glucosaminide(NP-GlcNAc) is hydrolysed by DspB and become 4-nitrophenol with the maximum light absorption at 405 nm. 20 mLbacteria solution is disrupted and acted supernatant as DsPb enzyme solution. We washed and shred the bacterial solution after induction, and took the supernatant as the enzyme reaction solution of DsPb while NP-GlcNAc working as a substrate.The enzyme activity experiment was carried out by using 100 μL of enzyme reaction solution and 100 μL of substrate solution (substrate concentration is 5 mM).

class=
Figure 4：DSPB enzyme activity assay curve

The enzyme activity of the supernatant calculated by the enzyme activity calculation formulais (Activity/(U/mL)=(kOD405-kseldecomposition)*18231.26 *dilution ratio) is 66.363U/mL.

The experiment proved that the recombinant bacteria expressed active DSPB, and we used the E. coli supernatant with DSPB activity to carry out the biofilm removal experiment.DH5a was cultured overnight in LB at 37 ° C and 220 rpm, and transferred to a 96-well plate at 37 ° C and cultured for 48 h, then discarded the supernatant .The biofilm was washed with PBS, and 200 uL of the reaction solution was added to react for the whole night. Add crystal violet to stain, wash the solution into the new well plate after alcohol washing, and measure the absorbance at 570 nm. The smaller the absorbance value, the better the membrane removal effect.

class=
Figure 4.(a) Crystal violet staining results of different reaction solutions added to biofilm (b) Biofilm removal rate of different reaction solutions added to biofilm (c) DSPB biofilm-removing curve OD570=51.96-51.24t0.0051 R2=0.98385

From Fig.5, The supernatant of the recombinant E. coli has the highest biofilm removal rate, and the biofilm removal effect is getting better with time.

https://static.igem.org/mediawiki/2018/0/00/T--ECUST--result--biofilm_remove_BIOFILM2.jpg