Part:BBa_K2737011
Plac-Dsp B-T7 terminator
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 1：The SDS of DSPB\ Lane 1,2 before induction
Lane 3 cell cultue media
Lane4,5,6 after induction
The expression of DspB was verified by SDS-PAGE(Figure1). The cell culture medium was blank control, and the pre-induction cell supernatant was used as a negative control. In the figure 1 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 uL of enzyme reaction solution and 100 uL of substrate solution (substrate concentration is 5 mM). File:T--ECUST--b3.2.tif Figure 12：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.
Figure 13.(a) Crystal violet staining results of different reaction solutions added to biofilm
(b) Biofilm removal rate of different reaction solutions added to biofilm The supernatant of the recombinant E. coli has the highest biofilm removal rate, and the biofilm removal effect is getting better with time..
(c) DSPB biofilm-removing curve
OD570=51.96-51.24t0.0051 R2=0.98385
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 231
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 264
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 49
Illegal SapI site found at 1309
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