Team:ECUST/Demonstrate

Demonstrate

CULTURE AND IDENTIFICATION OF IRON BACTERIA

Figure 1. (a)Bacteria is screened from rusty iron sample and cultured with Winogradsky culture medium. With rust and biofilm, bacterial colony is bronzing and circular. (b)Gram Straing：Gram-negative bacteria. (c)Iron bacteria with its biofilm (d)Electron microscope picture of iron bacteria with rust and biofilm complex.
Figure 2. The iron bacteria belongs to Pseudomonas sp. According to the identification results of 16s rDNA
Figure 3: Iron bacteria growth curve.Bacteria is cultured in Winogradsky culture medium at 30℃ and shake at 220rpm, 2% inoculum of culture solution (culturing age 3 days) and measure the light absorption at 600 nm.

QUORUM SENSING

We constructed a quorum-sensing system and found that the promoter with afe-box can work when the AHL concentration is more than 10-7 mol/L.

Figure 4. (a) Fluorescence intensity at different time induced by different concentrations of AHL. (b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate

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RUST REMOVER

We have not successfully constructed the recombinant plasmid with enterobactin because the gene cluster is too long.But we purified the enterobactin from E. coli successfully and tested the rust removal effect.

Figure 5. Fitting curves of oxalic acid and enterobactin (a) 3D fitting figure of rust removal rate for oxalic acid ;(b) 3D fitting figure of rust removal rate for enterobactin; (c) fitting curve of rust removal rate for oxalic acid ;(b) fitting curve of rust removal rate for enterobactin.

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BIOFILM REMOVER

We intesrt the gene of DispersinB (DSPB) to pET28a and E.coli expressed DSPB successfully. We performed enzyme activity assay and biofilm removal experiment by using cell supernatant. The enzyme activity of the supernatant is 66.363 U/mL. The supernatant has high biofilm removal rate, and the biofilm removal effect increased with time.

Figure 6. (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=0.98

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FUR INVERTER

The fur-box is inserted into three different positions of the promoter ,named Pfur,Pfur2 and Pfur.

Figure 7. (a) Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-5M (b)Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-7M. Fe excess medium is used to measure expression of report gene.

After adding with 10-7M Fe in medium ,Fur2 has the highest relative expression.So finally we choose fur2.

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LYSIN

Recombinant E. coli successfully expressed Lysin and can can make holes in the cell membrane.

Figure 8. The images are from scanning electron microscopes.(a)Before induction.(b)After induction.There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.

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BIOCIDE

Recombinant E. coli successfully expressed Lysin and we test the the sterilization effect of AD .

Figure 9. Growth curve of iron bacteria adding with different concentration of cecropin AD.

The figure shows that cecropin AD has powerful bacterividal activity. Only concentration of cecropin AD is low enough, can iron bacteria grow.

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LIGHT-ON SUICIDE

IRON BACTERIA-KILL CONPREHENSIVE CHARACTERIZATION