Team:TU Delft-Leiden/Project/Life science/curli/characterisation

Module Conductive Curli – Characterization

Plate Reader

A plate reader is a machine designed to handle samples on 6-1536 well format microtiter plates for the measuring of physical properties such as absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarisation.

The different constructs used are: p[rham]-CsgB – p[const.]–CsgA, also referred here as CC50, p[rham]-CsgB – p[const.]–CsgA:HIS, also referred here as CC51, p[rham]-CsgB-CsgA, also referred here as CC52, p[const.]-eGFP, also referred here as CC54

In this module, the cells carrying the curli-forming BioBricks (CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA), CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS),or CC52 (P[RHAM]-CSGB-CSGA)) also carried a plasmid constitutively expressing eGFP (CC54 (P[CONST.]-EGFP)). Hence, an assay to detect biofilm formation (due to the curli) can be performed. The cells can be grown on a 96-well plate, where curli formation will be induced with Rhamnose. The cells carrying CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA), CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS),or CC52 (P[RHAM]-CSGB-CSGA) together with CC54 (P[CONST.]-EGFP) will generate curli under these conditions, whereas the cells carrying CC54 (P[CONST.]-EGFP) alone will not. Under the Plate reader, the wells can be analysed for green fluorescence. Before washing out the cells all wells carrying cells with CC54 (P[CONST.]-EGFP) should present green fluorescence. After washing out the cells, however, only the wells carrying cells with CC54 (P[CONST.]-EGFP) together with one of the curli-forming BioBricks should still generate green fluorescence, because the curli would have made these cells attach to the walls of the wells and not being washed out. The final protocol developed for Plate reader analysis for this module can be found by clicking on this link .

Results - Plate Reader

Figure 1 shows the OD of the cells after two rounds of washing them out of the 96-well plate. On the image it can be appreciated that the cells carrying the curli-forming BioBricks (CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA)+CC54 (P[CONST.]-EGFP), CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS)+CC54 (P[CONST.]-EGFP) and CC52 (P[RHAM]-CSGB-CSGA)+CC54 (P[CONST.]-EGFP)) retain many more cells when they are induced with Rhamnose, whereas no noticeable increase of the OD is oserved under induction for the cells that do not carry curli-forming constructs (CC54 (P[CONST.]-EGFP) alone and empty cell). This suggests that cell retention happens when the curli genes are expressed.

Confocal Microscopy

Confocal microscopy is an imaging technique that allows for the visualisation of fluorescent bodies with higher resolution and improved contrast compared to Bright-field microscopy. Whereas fluorescent Bright-field microscopes excite all the sample analysed, confocal microscopes can highly reduce the excited field, thus eliminating the background noise produced by species neighbouring the body of interest.

We used confocal microscpoy technology to observe the deposition of cells at the bottom of the microscope slide. Figures 2-6 intend to represent how after induction with Rhamnose the cells forming curli are attached faster to the surface (bottom) of the microscope slide than when they are not induced.

The fact that more cells are observed at the bottom for the ones carrying the CC54 (P[CONST.]-EGFP) plasmid alone, or the empty cells is attributed to the fact that these cells grow faster because they do not have the burden of carrying an extra plasmid, or even two in the case of the empy cells. This idea is supported by the fact that the induction of the curli-forming genes clearly indicates a faster deposition of the cells onto the surface of the microscope slide.

Congo Red Assay

We did a Congo Red assay on the following cell cultures: CC54 (P[CONST.]-EGFP) + CC52 (P[RHAM]-CSGB-CSGA), CC54 (P[CONST.]-EGFP) + CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA), CC54 (P[CONST.]-EGFP) + CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS) and the used strain without plasmid. The protocol that was used for the assay can be found here. We took samples spread over two days and did the following for each sample: first measured the OD₆₀₀ to be able to correct for growth. Then added Congo Red, waited for five minutes and measured the OD₄₈₀. If curli (biofilm) is formed, the Congo Red dye will get stuck in the curli biofilm and therefore will be stuck in the pellet after centrifugation. Of course the difference between the non-induced cultures and the induced cultures are the most important, therefore the comparison between the induced and non-induced samples. The results of our assay can be found in figure 7.

In figure 7 we can see that the samples with induced CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA), CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS) and CC52 (P[RHAM]-CSGB-CSGA) result in a higher value than the non-induced samples, meaning that the OD₄₈₀ values are more negative (as we measured these in negative values). From this we can deduct that more Congo Red dye got stuck in the pellet (also see figure 8 and 9) in the CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA) induced, CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS) induced and CC52 (P[RHAM]-CSGB-CSGA) induced cultures and therefore more biofilm was formed. The negative control of empty cells gives roughly the same value for induced as for non-induced cells, which points to the same amount of curli produced. Together with the results that the empty cells have around the same value for the -OD₄₈₀ divided by the OD₆₀₀ as the non-induced CC50, CC51 and CC52, this results in the conclusion that the empty cells do not produce curli.

It should be noted that we only took two measurements the first day and left the cultures overnight before conducting the last measurement. As part of the collaboration, Wageningen repeated the Congo Red assay and these results can be found in the following paragraph.

In figure 10 we can see the results of the Congo Red assay Wageningen performed for us as part of the collaboration. They used the same protocol as we did, it can be found here. The results are partially the same, except for the large negative value for CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS). (Explanation!) Wageningen also measured over two days, just as we did. But because they had more measurements, we could also make a OD₄₈₀ versus time plot (figure 11).

From this timeplot we can see that the OD480 increases over time... (Do we actually want this plot in the text or is it not an addition to the information?)

Crystal Violet assay and Gold Nanoparticle

The Congo red experiments prove that after induction with rhamnose the curli-proteins CsgA and CsgB were produced. This does however not prove that bio-film is formed; to prove bio-film formation a crystal violet assay was performed together with the experiment for conductance. Crystal violet (or Methyl violet) is an organic dye that is used in Gram-staining to colour the cell-wall of bacteria [1] The protocol for the assay can be found here[LINK!!!!!!!!!!]. Bacteria are grown in petri-dishes with liquid medium without shaking, thereby allowing them to create a layer of biofilm on the surface of the plastic. After incubation the petri-dishes are emptied and submerged in MQ to wash away any non-bound cells. Bacteria were now incubated with Crystal violet dye, which colours them purple. Then the biofilm was resuspended in acetic acid and the absorption at 590nm, the absorption peak of crystal violet, was measured. Figure 11 shows a picture of the biofilm in the plates containing BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst) that were induced. The results of the absorbance measurements can be seen in table 1.

Mother Machine - Widefield Fluorescence Microscopy

A Widefield Fluorescence Microscope was used to characterise the the eGFP reporter BBa_k1316016 in the Mother Machine (MM), which was used as a positive control Curli module. For more information about the Mother Machine, please visit our Microfluidics section.

MM Devices were flushed with Bovine Serum Albumin (BSA) to render the PDMS out of which the MM is made inert after plasma activation. Then cells grown in M4 minimal medium supplemented with 40mM glucose were flowed through. The M4 medium is used as a growth medium because it does not exert autofluoresence and the diameter of the cells are smaller as compared to those grown in rich media; a small diameter is required for the cells to fit in the side-channels of the MM.

The devices were then centrifuged at 3000rpm for 10 minutes, with side channels of the MM in the direction of the centripetal force. In order to coax the cells into the small channels on one side.

Unfortunately, individual cells were not found in the side channels. Reasons for this are unclear, possible causes are faulty or damaged moulds, or human error in the fabrication process. However, cells could still be imaged in the main channel, and characterised for flouresence.

Conclusions

From the assays performed it can be concluded that:

The constructs made are capable of generating curli-forming proteins

Curli formation happens in response to induction of the used promoter (induced with the presence of Rhamnose)

BioBricks CC50 (P[RHAM]-CSGB – P[CONST.]–CSGA) and CC51 (P[RHAM]-CSGB – P[CONST.]–CSGA:HIS) do not show a clear improvement compared to CC52 (P[RHAM]-CSGB-CSGA). Consequently, contrary to what we expected, constitutively express CsgA protein does not seem to speed up the nucleation process of curli formation with the constructs used.