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

Module Conductive Curli – Characterization

In the wet lab we made constructs containing csgA and csgB, two of the genes involved in curli formation. Here you can find information with respect to the characterization of the BioBricks for the Conductive Curli pathway.

The different constructs made for this module are:

• BBa_K1316013: p[rham]-CsgB – p[const.]–CsgA, also referred here as CC50
• BBa_K1316014: p[rham]-CsgB – p[const.]–CsgA:HIS, also referred here as CC51
• BBa_K1316015: p[rham]-CsgB-CsgA, also referred here as CC52
• BBa_K1316016: p[const.]-eGFP, also referred here as CC54

The strains used to characterize these constructs contain a combination of a curli-forming BioBrick (CC50, CC51 or CC52) plus the construct constitutively expressing eGFP (CC54). As negative controls, a strain containing the constitutively expressed eGFP alone (CC54) and an empty strain (containing no constructs) were used.

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 (CC50, CC51 or CC52) also carried a plasmid constitutively expressing eGFP (CC54). 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 L-Rhamnose. The cells carrying CC50, CC51 or CC52 together with CC54 will generate curli under these conditions, whereas the cells carrying CC54 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 should present green fluorescence. After washing out the cells, however, only the wells carrying cells with CC54 together with one of the curli-forming BioBricks should still generate green fluorescence, because of cell attachment to the walls. 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 + CC54, CC51 + CC54 and CC52 + CC54) retain many more cells when they are induced with L-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 cells). 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 L-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 of the microscope slide for the strains carrying the CC54 plasmid alone, or the empty cells could be 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 strains carrying curli-forming constructs (CC50, CC51 or CC52) seem to be deposited faster onto the surface of the microscope slide when they are induced than when they are not.

In order to do an objective evaluation of the number of cells, the total intensity of fluorescence of the imaged microscope slides can be done. If the fluorescence per cell of all the different strains is the same, then the more fluorescence on the microscope slide, the more cells would be attached on the surface. Then, the fluorescence per OD for each working strain was determined using the data of OD and fluorescence already available from the plate reader (the data after the 2nd cell wash was used). Figure 7 shows that all cells present the same fluorescence per OD, which makes sense as eGFP is constitutively expressed in all of them under the regulation of the same promoter. The only strain that does not make sense is the empty strain, because it is supposed to have no BioBrick and, therefore should not emit fluorescence. A possible reason for this result is the fact that LB medium was used for this assay, and it is auto-fluorescent. Another reason that could explain this is the fact that transparent 96-well plates were used for the assay. Although this well plates are supposed to be suitable for fluorescence measurements, the fact that the cells that produced fluorescence after 2 rounds of washing were, in principle, attached to the walls of the well plates could have produced noise in the measurement of neighbouring wells (producing, for instance, fluorescence noise on the wells of the empy cells). The fact that in the confocal microscope no fluorescent cells were observed supports the idea that something might have gone wrong in the measurement of fluorescence of these empty cells.

Once fluorescence per OD was measured, the total fluorescent intensity was measured per microscope slide using the ImageJ software (figure 8). According to this data, CC50 is the construct that shows more cell attachment to the slide bottom when csgA protein is induced in relation to the same non-induced strain. CC52 shows a bigger attachment improvement when induced than the CC51 construct.

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. 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 proteins are formed, the Congo Red dye will get stuck in the curli 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 9.

In figure 7 we can see that the samples with induced CC50 and CC52 result in a higher value than the non-induced samples. 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. The more dye in the pellet, the more curli has been produced. 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 produce a low amount of or no 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 a collaboration, Wageningen iGEM 2014 repeated the Congo Red assay and these results can be found in the following paragraph.

In figure 11 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 induced samples give approximately the same value for the ratio –OD₄₈₀ divided by OD₆₀₀ as the induced samples in our own measurement. This confirms our results and with that our conclusions. Wageningen also measured over two days, just as we did, but because they had more measurements, we could also make an OD₄₈₀ versus time plot (figure 12).

From this timeplot we can see that the OD480 increases over time and that the curli production already starts about 4 hours after induction.

From both datasets we can conclude that curli is being produced in the cells, but we did not see any difference between CC50 + CC54, CC51 + CC54 and CC52 + CC54, while we had expected to see a higher nucleation speed for the constitutively expressed CsgA protein (constructs CC50 and CC51).

Crystal Violet assay and conductance measurements

Crystal Violet assay

The Congo red experiments prove that after induction with L-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 . E.coli ΔCsgB bearing the BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst) are grown in petri-dishes with liquid medium without shaking. When induced this allows them to create a layer of biofilm on the surface of the plastic. After incubation the petri-dishes were emptied and submerged in MQ to wash away any non-bound cells. Bacteria were now incubated with Crystal violet dye, which colours them purple. The biofilm was then resuspended in acetic acid and the absorbance at 590nm, the absorption peak of crystal violet, was measured. Figure 13 shows a picture of stained biofilm in dishes containing BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst) that were induced. The results of the absorbance measurements can be seen in table 1.

Table 1: Absorbance measurements at 590nm performed on E.coli ΔCsgB with BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst) and a negative control with untransformed cells after the crystal violet assay.

Exp. #	Strain	Biobrick	Induced	A590
1.	E.coli ΔCsgB	BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst)	Yes	1.256
2.	E.coli ΔCsgB	BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst)	Yes	1.066
3.	E.coli ΔCsgB	BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst)	No	0.310
4.	E.coli ΔCsgB	-	No	0.082

Table 1 quantitatively shows that when BBa_K1316014 was induced with L-Rhamnose that biofilm was formed, whereas the negative control which was untransformed E.coli ΔCsgB showed marginal biofilm formation. Experiment 1 and 2 seem duplicate of one and other, however this is not the case since Gold nanoparticles were added to experiment 4 as will be elaborated on in the conductivity measurement part further down. These gold nano-particles might also absorb at 590nm therefore yielding a higher absorbance in exp.# 4. It is peculiar the uninduced strain bearing the biobrick also shows some biofilm formation. This might be due to the fact that the L-Rhamnose-promoter is leaky or alternatively that it is induced by other carbon-sources in the medium like glucose.

Conductance measurements

Proteins containing a His-tag can bind Cu and Ni in a coordinance bond. Our BBa_K1316014 (CsgB + pRha, CsgA-His+ pConst) biobrick has a His-tag, and can therefore bind Gold nanoparticles (GNP) that are attached to a Ni atom via a NitriloTriacetic Acid (NTA) chain. CsgA forms curli fibrils that are attached to the cell via CsgB; curli is a fibril that is abundant in the extracellular matrix in biofilm. Because our CsgA has a His-tag it can bind gold-nanoparticles thereby facilitating the conductance of electricity through biofilm. We assayed the conductance of biofilm with and without GNPs on Dropsens integrated electrodes as shown in figures 14 A and B. The protocol for conductance measurements can be found here . A schematic of the interaction between a His-tag and the Ni-atom on the GNPs is represented in figure 14C.

The resistance was measured with a conventional multimeter with a maximum resistance of 20MOhm. Both the biofilm with and without GNPs had a resistance of over 20MOhm.

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 (figure 15).

Conclusions

From the assays performed it can be concluded that:

• The constructs made are capable of generating curli-forming proteins and biofilm as proved by Congo red and crystal violet assays respectively.
• Curli formation happens in response to induction of the used promoter (induced with the presence of L-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.
• We could not prove that biofilm to which Gold nano-particles were bound was more conducting than without the nano-particles due to the maximum resistance measured by our multimeter.