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

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Curli Module - Characterisation

Assays

The different assays used to test our Curli BioBricks are:

Plate Reader

Confocal Microscopy

Congo Red Assay

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.

In this module, the cells carrying the curli-forming BioBricks (BBa_K1316013-BBa_K1316015) also carried a plasmid constitutively expressing eGFP (BBa_K1316016). 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 BBa_K1316013-BBa_K1316015 together with BBa_K1316016 will generate curli under these conditions, whereas the cells carrying BBa_K1316016 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 BBa_K1316016 should present green fluorescence. After washing out the cells, however, only the wells carrying cells with BBa_K1316016 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.

LINK TO PROTOCOLS

Results - Plate Reader

Figure 1: OD after washing out the cells twice as a fraction of initial OD observed on 96-well plates, with (+) and without (-) induction of the curli-formation genes. Induced cells are induced with 1% rhamnose solution.

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+CC54, CC51+CC54 and CC52+CC54) 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 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 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.

Figure 2: Fluorescent images taken using the Confocal Microscope of the cells carrying the CC constructs CC50 and CC54, induced (left) and non-induced (right).

Figure 3: Fluorescent images taken using the Confocal Microscope of the cells carrying the CC constructs CC51 and CC54, induced (left) and non-induced (right).

Figure 4: Fluorescent images taken using the Confocal Microscope of the cells carrying the CC constructs CC52 and CC54, induced (left) and non-induced (right).

Figure 5: Fluorescent images taken using the Confocal Microscope of the cells only carrying the CC construct CC54, induced (left) and non-induced (right).

Figure 6: Fluorescent images taken using the Confocal Microscope of the empty cells carrying no CC construct fluorescence mode(left) and Bright-field mode (right).

Congo Red Assay

We did a Congo Red assay on the following cell cultures: CC54 + CC52, CC54 + CC50, CC54 + CC51 and the used strain without plasmid. The protocol that was used for the assay can be found here. (LINK THIS) We took samples every hour and did the following for each sample: First measured the OD₆₀₀ to be able to correct for growth. Then added Congo Red, waited for a while 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, CC51 and CC52 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 in the CC50 induced, CC51 induced and CC52 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.