Part:BBa_K1404006

p70-CsgA, curli generator

This part combines the short curli promoter P70 (BBa_K342000) with a wild-type csgA gene, allowing to produce the main curli subunit, CsgA. In the presence of the rest of the Curli machinery (CsgB/E/F/G), this allows E. coli to form biofilms. CsgA can also capture small quantities of nickel.

Characterization

Adherence

Using confocal microscopy, we showed that the part K1404006 does not modify the adherence of a naturally curli-producing strain. Moreover, quantitative adherence tests showed that this part can successfully complement a CsgA- strain.

For the Confocal Laser Scanning Microscopy biofilm acquisitions, all the strains were cultivated in 96-wells microplate in M63 Mannitol during 16 h at 30°C (See the complete protocol for details). See results in Figure 1.

Figure 1: Engineered bacteria biofilm characterization and quantification using Confocal Laser Scanning Microscopy

All the strains used are constitutively fluorescent to allow detection with confocal laser microscopy (ZEISS LSM510 META, 40X/1.3OILDIC, laser Argon 4 lines 30 W 458 nm, 477 nm, 488 nm, 514 nm, See Protocols). Positive control/CsgA+ (Wild-type E.coli curli producing strain); Negative control/CsgA- (csgA-knockout E.coli strain); BBa_CsgA (BBa_K1404006); BBa_CsgAHis1 (BBa_K1404007); BBa_CsgAHis2 (BBa_K1404008). A) Biofilm sections obtained by Z-stack acquisitions. B) Biofilm 3D reconstruction using IMARIS® from acquisitions in A). C) Bio-volume quantification and maximum of thickness measurement using COMSTAT2 (ImageJ). The strain marked with a star is significantly different from all others (Tukey’s test, p<0.05).

As the strains carrying BBa_K1404006 does not show a significant difference with the positive control, this parts insertion doesn’t modify the biofilm formation property.

Nickel capture

Despite not having any specific Nickel-binding motifs, the part K1404006 can chelate small amounts of nickel.

Nickel(II) chelation was evaluated in a CsgA- MG1655 background (in order to have only our modified or unmodified curlis at the surface of the strain) for each of the constructions (BBa_K1404006, BBa_K1404007, or BBa_K1404008). Dimethylglyoxime (DMG) was used as a complexing reagent, which forms a pink-colored complex (peak absorption at 554 nm) in the presence of Ni(II).

Firstly, a calibration curve of the formation Nickel and DMG complexes was established.

Then, strains were assayed for biofilm nickel absorption on liquid cultures using the calibration curve, by measuring the OD of the complex formed for each strain at 554 nm.
Although quantification is possible, this technique lacks precision and is more suited for qualitative studies. However, it is a cheaper alternative to mass spectrometry.

Nickel-DMG complex colorimetry measurement follows a linear regression from a concentration of 20 µM to 100 µM, linked to the gradient from transparency (at 20 µM) to pink (at 100 µM). This visual method allows us to compare the Ni chelation between our strains. The more pale the color is, the more Ni has been chelated. The culture supernatant (=remaining nickel) of CsgA- bacteria from strain with the part BBa_K1404008 is less colored than the others, which shows that only this part allows to capture more nickel. These results show that It is shown that it chelates more than part BBa_K1404006 and part BBa_K1404007.

A second method has been used, more quantitative and more precise (but more expensive) : mass spectrometry . The metal content of the bacterial pellets were assayed. The quantity of chelated nickel for each strain has been compared to the quantity of curlis formed by each strain.

Significant differences are indicated using lowercase letters, and different letters indicate significant differences (Tukey’s test, p < 0.05). Error bars represent standard deviations.

Taken together, these results show that the CsgA- Strain with part BBa_K1404008 chelates twice more than strain CsgA- with part BBa_K1404007. That means that only two His-tags on C-term can improve the natural nickel chelation capacities of CsgA . CsgA with a single His-tag did not perform better than a wild-type CsgA. Potentially, further increasing the amount of His-tags could improve the nickel accumulation capacities of CsgA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]