Part:BBa_K1404008

p70-CsgA-His*2, double His-tagged curli generator

This part combines the short curli promoter P70 (BBa_K342000) with a double poly-His-tagged (6X His-Linker-6X His) csgA gene, allowing to produce the main curli subunit, CsgA, with two His-tags. In the presence of the rest of the Curli machinery (CsgB/E/F/G), this allows E. coli to form biofilms. CsgA-His2 can also capture large quantities of nickel.

Characterization

Quantitative adherence tests showed that this part can successfully complement a CsgA- strain. Using confocal microscopy, we demonstrated that the part K1404008 does not modify the adherence of a naturally curli-producing strain.

Thanks to its specific Nickel-binding motif, the part K1404008 chelates more nickel than a wild-type CsgA.

Adherence

Five complementary tests were performed to evaluate the ability of the modified cells to assemble functional curli:
1) determination of the percentage of adherent cells to polystyrene in 24 wells-plates,
2) crystal violet staining of biofilm formed on polystyrene in 24 wells-plates,
3) ability to bind the congo red,
4) biofilm maximum thickness measurement and biovolumes quantification of GFP-tagged biofilm observed with a confocal microscopy and
5) curli structure observation using Transmission Electron Microscopy (MET).

Adhesion test and curli production

Figure 1 : Engineered bacteria Percentage of adhesion

csgA-knockout E. coli strain was transformed with BBa_CsgA-WT (BBa_K1404006); BBa_CsgA-His1 (BBa_K1404007); BBa_CsgA-His2 (BBa_K1404008). The corresponding positive and negative controls are Wild-type E.coli MG1655 curli producing strain transformed with the empty vector and csgA-knockout E. coli strain transformed with the empty vector, respectively.
Strains with our parts, the positive and negative controls were cultivated in 24-wells microplate in M63 Mannitol during 24H at 30°C. The supernatant was removed and the OD₆₀₀ measured, then the bacteria forming the biofilm were resuspended and the OD₆₀₀ measured in order to estimate the number of cells (See protocol for details ). The percentage of adhesion was calculated as follow : (OD₆₀₀ of the biofilm)/ (OD₆₀₀ of the supernatant + OD₆₀₀ of the biofilm)
Different uppercase letters displayed on the graph indicate significant differences between strains (Tukey’s test, p < 0.05)

These results show that the percentage of adhesion is similar between the strains containing K1404008 and the positive control, thus the His-tagged CsgA is functional.

Figure 2 : Engineered bacteria Biofilm formation

The cells were grown as described in figure 1.

The supernatant was removed and the remaining biofilm was fixed to the microplate by heat treatment at 80°C during 1 h. The crystal violet solution was added in each well in order to stain the cells and the wells were washed with water to remove crystal violet in excess (See protocol for details ).

Crystal violet staining shows that the strain containing K1404008 could form a biofilm like the positive control, thus the tagged CsgA is functional.

Figure 3 : Engineered bacteria curli production

Strains are the same as in figure 1.
Strains with our parts, the positive and negative control were cultivated in M63 Mannitol at 30°C and 180 rpm. After centrifugation, the supernatant was removed and the cell pellet was resuspended in the Congo Red solution, in order to specifically stain the curli. The samples were centrifuged again and the pellets observed (See protocol for more details).

Congo Red staining shows that the CsgA-His produced from this part allows to form curli fibers which are able to bind Congo Red.

Confocal microscopy

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 4.

Figure 4: 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_K1404008 does not show a significant difference with the positive control, this parts insertion doesn’t modify the biofilm formation property.

Transmission Electron Microscopy

For the Transmission Electron Microscopy, all the strains were cultivated in conditions that allow the formation of the curli : 48h (for a 50 mL culture), 28⁰C temperature and low agitation. The ammonium molybdate was used as a negative colorant (Microscope: MET PHILIPS CM120).

Figure 5: Engineered bacteria curli structure observation using Transmission Electron Microscopy

The bacteria cultures we used are a csgA-knockout strain as negative control, and the three engineered csgA constructions, the WT, the His1-Tag and His2-Tag.

The images show that there is no significant difference between our positive control and our constructions. Thus, we can conclude that our parts insertions don’t affect the structure of the amyloid fibers and the configuration of the curli formation.

Nickel capture

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 one of the constructions (BBa_CsgA-WT (BBa_K1404006), BBa_CsgA-His1 (BBa_K1404007),or BBa_CsgA-His2 (BBa_K1404008)). Dimethylglyoxime (DMG) was used as a complexing reagent, which forms a pink-colored complex (peak absorption at 554nm) in the presence of Ni(II).

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

Then, liquid cultured strains were assayed for biofilm nickel absorption using the calibration curve, after measuring the OD of the complex formed for each strain at 554nm. (See Protocol for details)
Although quantification is possible, this technique lacks precision and is more suited for qualitative studies. However, it is a cheaper alternative to ICP-MS.

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 prove that the part BBa_K1404008 confers increased chelation to strain CsgA-. 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) : inductively coupled plasma mass spectrometry (ICP-MS). 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.

Different lowercase letters displayed on the graph indicate significant differences between strains (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 (K1404007) 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]