Background

In the kinetic experiment, the fluorescence signal of the proteins CsgA and CsgA-GFPmut3 (I13504) proteins expressed by our constructs was recorded in time. Besides fluorescence, we measured the OD600 of our cultures in order to normalize the fluorescence signal per cell. The main goal of this experiment was to see, if different induction levels with increasing rhamnose concentrations would lead to a higher production of CsgA. If this was true, the quantity of produced CsgA present in the medium could be controlled. This degree of control is key to achieving our main goal; making a biofilm with reproducible strength.

The obtained results can be used to calculate the promoter strength at different induction levels of rhamnose in our model. In order to use the mathematical model previously constructed in the modeling section, it has to be fitted to GFPmut3 units/cell/second. Therefore, we set up a calibration curve with exactly the same settings as the fluorescent experiment in order to correlate fluorescence signal to units of GFPmut3 per cell.

Methodology

In this experiment, the strains ∆csgA – csgA- I13504 and ∆csgA – csgA strain were both induced with rhamnose at different concentrations. The different induction levels can be found in Table 1.

All conditions (ID 1 – 6) were carried out in triplicates for accurate statistical analysis of the data. The different cultures were grown and induced in a 96-well plate. The OD600 and the fluorescence signal was recorded with a plate reader during an 18-hour period at 30°C.

Results and discussion

In Figure 5, the fluorescent signal was normalized with the number of cells and plotted as a function of time. The red bars denote the error within each ID.

As observed in Figure 5, only the strains carrying the csgA-GFPmut3 construct induced with 0.2% (w/v) and 0.5% (w/v) showed a clear increase in fluorescence signal over time. The rest of the cultures, didn’t show significant fluorescence over time.

Furthermore, we have showed that increasing concentrations of rhamnose lead to increasing amounts of produced csgA-GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active directly after induction, but activity is observed after a time period of 3 to 4 hours. This is in accordance with data from literature (Wegerer et. Al, 2008), in which low fluorescence levels were observed after 2 hours of induction of the rhamnose promoter.

The calibration line of fluorescence versus mass amount GFPmut3 is given in Figure 6.

The corresponding function of the GFPmut3 calibration line is:

With mass_GFP in ng.

In the modelling, the fluorescent data in Figure 5 will be further converted to molecules GFPmut3/cell and the promoter activity will be calculated for both the 0.2% (w/v) and 0.5% (w/v) level of rhamnose induction. With this kinetic experiment, we have proven that our csgA-GFPmut3 construct is able to produce different levels of GFPmut3 by varying the rhamnose concentration.

Background

With our biofilm formation model we aimed to estimate how many times the nanowires overlap in order to predict the biofilm strength. One of the variables required for this model is the persistence length. In order to determine it, we have to visualize the nanowires produced by our engineered bacteria. Moreover we wanted to visualize the difference between induced and uninduced cultures containing the csgA_pSB1C3 plasmid with a rhamnose inducible promoter. These experiments required using an electron microscope.

Electron microscopes use electrons to visualize nano-objects, such as the curli nanowires. In the large “gun” the electrons get enough space to accelerate before they illuminate the sample. As shown in Figure 7 after the electrons are fired by the gun, an electric field of several thousand volts is applied to the electron beam in order to point this to the sample. In the case of an TEM, the electrons pass through the sample where the detector can detect the sample (Boysen & Muir, 2011).

Methodology

At first we measured an OD600 of 3.00 of an overnight culture of ∆csgA_csgA_pSB1C3 cells. We diluted the sample down to an OD600 of approximately 0.05, with LB medium + CAM (antibiotic, chloramphenicol). To achieve an end volume of 4 mL we took 67 µl of the overnight culture and 3933 µl of LB + CAM. We let the cells in the new sample grow for 1.5 hour at 37°C to reach the exponential phase (OD between 0.35-0.60). After the cells reached the exponential phase, we induced 1 mL of the culture with 1% rhamnose (250 µl from our 5% rhamnose stock) and also took 1 mL sample of the uninduced culture. We incubated both samples overnight at room temperature to allow the production of nanowires. Finally, the samples were directly added to the grid and the following TEM pictures were taken.

Results

In Figure 8, both the situation, pictured with TEM, without induction (the left one) and with induction (the right one) are shown with a magnification of 7300 x. The situation in which there was no induction with rhamnose, the bacteria did not show any curli formation. The opposite was true for the case in which the cells were induced with rhamnose. As the TEM picture clearly shows, nanowires are present after induction.

Background

The bacteria that we engineered for the project are capable of producing curli proteins after induction with L-rhamnose. However, the goal of our project is the printing of biofilms and not the sole overexpression of these proteins. In order to assess whether our CsgA-producing bacteria can make a biofilm or just remains planktonic, our team adapted the protocol from Zhou et al. (2013) that employs crystal violet (methyl violet 10B) for dying the biofilm-making bacteria that attach to surfaces.

According to the source article (Zhou et al., 2013), a deficient mutant of Escherichia coli is unable to produce a biofilm. By performing this experiment, we want to go further in characterizing our curli proteins; we want to answer this question: are we really making biofilms?

Methodology

In the experiment, our CsgA-producing strain of E.coli is induced at a high (0.5% w/v), low (0.2% w/v) and no (0% w/v) concentration of L-rhamnose. Furthermore, csgA deficient bacteria transformed with an empty plasmid (pSB1C3) are also tested and used as a control (Table 2.). All the experiments were carried out in triplicates.

The cultures of both strains were grown and induced in a 96-well plate. 40 hours after the induction, the samples were washed with water (in order to disregard non-attached, planktonic cells) and dyed with crystal violet (figure 9.).

Results and discussion

In the end, the wells were diluted with ethanol so all the content can dissolve in the liquid phase. We measured the absorbance at 590 nm of wavelength for all the samples, obtaining the following results (figure 10.):

The cells induced with l-rhamnose showed an increased absorbance at 590 (the peak of the dye) when compared to the empty-plasmid controls, and also with the non-induced sample. That results clearly demonstrate how our engineered cells with BBa_K1583000 successfully make biofilms, regarding that the empty plasmid controls and the analysed samples have significantly (table 3.) higher crystal violet retention. On the other hand, a higher concentration of rhamnose is not leading to a higher expression. The cells induced with a 0.2% w/v of rhamnose seem to create better a biofilm structure. However, this could be a consequence of different growth patterns; the cultures induced with 0.5% w/v of rhamnose could have stopped duplicating earlier, so the cell concentration could have decreased. So, the dyed area could be consequently smaller.

The cultures of both strains were grown and induced in a 96-well plate. 40 hours after the induction, the samples were washed with water (in order to disregard non-attached, planktonic cells) and dyed with crystal violet (figure 9.).

Results and discussion

In the end, the wells were diluted with ethanol so all the content can dissolve in the liquid phase. We measured the absorbance at 590 nm of wavelength for all the samples, obtaining the following results (figure 10.):

The cells induced with l-rhamnose showed an increased absorbance at 590 (the peak of the dye) when compared to the empty-plasmid controls, and also with the non-induced sample. That results clearly demonstrate how our engineered cells with BBa_K1583000 successfully make biofilms, regarding that the empty plasmid controls and the analysed samples have significantly (table 3.) higher crystal violet retention. On the other hand, a higher concentration of rhamnose is not leading to a higher expression. The cells induced with a 0.2% w/v of rhamnose seem to create better a biofilm structure. However, this could be a consequence of different growth patterns; the cultures induced with 0.5% w/v of rhamnose could have stopped duplicating earlier, so the cell concentration could have decreased. So, the dyed area could be consequently smaller.