Part:BBa_K1583104

pRha + Mfp5_CsgA_His fusion protein

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. Inspired by mussels, the Mfp5 (mussel foot protein) has high adhesive properties towards wet polar surfaces. By creating a fusion protein, the adhesive properties of the mussel foot protein is combined with the formation of nanowires.

The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al, 2014).

</html> Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 245
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Four different experiments were done to characterise the BBa_K1583104 biobrick:

• Fluorescence assay
• Crystal Violet assay
• Western blot
• Transmission electron microscopy

Fluorescence assay

To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (BBa_I13504) under induction of the same rhamnose promoter (BBa_K1583112) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C.

In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID.

Fig. 1: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments.

As can be seen from Fig. 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with the csgA-GFPmut3 construct gave a clear increase in fluorescence signal in time. All other experiments, gave similar levels of fluorescence, slightly increasing in time. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in 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 right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.

With this kinetic experiment, we have proven that the rhamnose promoter does indeed induce the expression of the csgA gene.

Crystal violet assay

The assay above showed that the bacteria that we engineered for the project is capable of producing the CsgA proteins after induction with L-rhamnose. However, this did not yet prove that curli are formed. In order to assess whether our bacteria producting CsgA with a His tag can still produce these nanowires, our team adapted the protocol from Zhou et al. (2013) that employs crystal violet (methyl violet 10B) for dying the biofilm-making bacteria that attaches to the surface. In the experiment, our CsgA_His-producing strain of E. coli was 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) were used as control. 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 3.).

Figure 3. Microtiter Plate Assay results for testing biofilm formation. All the measurements were conducted in triplicates. CTRL0, CTRL2 and CTRL5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with pSB1C3, used as a control. MFP30, MFP32 and MFP35 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains csgA attached to the mfp3 peptide under an inducible promoter. The termination 0, 2 and 5 denote the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).

As we also observed in the crystal violet assay for the plasmid containing the csgA gene, the sample that can also express RFP as a reporter gene is also able to make a biofilm. That confirms that this strain can be used for the experiments of characterization of the 3D printing, as the reporter gene expression is not interfering in the biofilm-making ability.

On the other hand, the tagged CsgA proteins seem to have biofilm-making capability despite having a peptidic modification in the structure. To confirm that there is a real change between the analysed samples and the empty plasmid control, a significance analysis was performed for α=0.05 (Table 8.).

Table 1. Significance analysis of the samples analysed, with a significance α value of 5%. All the samples but the 0.5% induction of CSGARFP5 display a significant difference when compared to the empty plasmid homolog sample (CTRL).

The significance analysis shows that cells containing the Mfp5_CsgA_His (BBa_K1583104) biobrick can efficiently create a curli, when compared with an empty plasmid control (i.e. without csgA expression).

Western blot

For our modeling, we needed to determine the internal concentration of csgA. Based on the fluorescent assay with GFPmut3, we obtained an internal amount of CsgA in the order of 104 molecules/cell. To validate whether this value was plausible we decided to use Western Bloting. The following strains were used (Table 2.) (ΔcsgA is a csgA deficient strain called E. coli K-12 MG1655 PRO ΔcsgA ompR234).

Table 2: Samples used for the first Western Blot experiment.

Sample #	Description	Time of induction (hours)
1	ΔcsgA - csgA	5
2	ΔcsgA - csgA + Rhamnose	5
3	ΔcsgA - csgA_His	5
4	ΔcsgA - csgA_his + Rhamnose	5
5	ΔcsgA - csgA	Overnight
6	ΔcsgA - csgA + Rhamnose	Overnight
7	ΔcsgA - csgA_his	Overnight
8	ΔcsgA - csgA_his + Rhamnose	Overnight

The procedure of this experiment is described here at Protocols > "Isolation using Ni-column purification assay (QIAGEN kit) for intracellular protein tagged with 6xHis³".

The SDS gel is shown in figure 4. The gel is too condensed to visualize a difference between the non-induced and induced samples, because the gel was overloaded.

Figure 4. Left: SDS page gel, experiment 1 samples as described in Table 2. Rigth: Membrane after blotting, of the samples of experiment 1

We noticed in figure 4 a band in lane 4 and 8, with a size of circa. 17 kDa. The lanes correspond to the rhamnose induced strain E. coli K-12 MG1655 PRO ΔcsgA ompR234 (ΔcsgA) with biobrick BBa_K1583102 (CsgA_His), after 5 hours and 24 hours of induction. The antibodies bind specifically to the his-tag of proteins, which did not bind to the ΔcsgA_csgA samples, lane 1, 2,5 and 6. Therefore it can be concluded that the strain ΔcsgA_csgA_his produced his-tagged CsgA proteins. The effect of rhamnose was visible when compared to the samples with ΔcsgA_csgA_his without rhamnose induction, lane 3 and 7. There are no bands visible, therefore rhamnose induces the production of CsgA_His. We can conclude with experiment 1 that the antibodies bind specifically to CsgA_His proteins. The CsgA_His proteins are only present when induced with 0.5% rhamnose.

We executed a second experiment to quantification the amount of CsgA_His present in the ΔcsgA_csgA strain. We compared this amount by using known concentration of CsgA from a strain called T-SSRA madrid. T-SSRA madrid strain has a set amount of CsgA_His proteins present in the cell.

Table 3: Samples used for the second Western Blot experiment.

Sample #	Description	Time of induction (hours)
1	ΔcsgA - csgA_his + Rhamnose	5
2	ΔcsgA - csgA_his + Rhamnose	Overnight
3	T-SSRA madrid 20µM	/
4	T-SSRA madrid 8µM	/
5	T-SSRA madrid 4µM	/
6	T-SSRA madrid 2µM	/
7	T-SSRA madrid 0.4µM	/
8	T-SSRA madrid 0.2µM	/

The same protocol as in the first western blot experiment (above) was used.

Figure 5. Membrane after western blotting; samples of experiment 2.

As seen in figure 5, the bands of the calibration curve were visible. The bands of the ΔcsgA_csgA_His were not visible. We still wanted to know the correlation between the thickness of the band, and the concentration of internal CsgA_His. We solved this by comparing the band in lane 8 from figure 5 with the bands seen in figure 4, to acquire an approximation of the total concentration. The bands in lane 4, and 5 (right to left) were too saturated to measure with the Typhoon. The band in figure 4 was thinner than the two highest concentrations, therefore we removed the last two points from the calibration line. The trend line of the concentration (in µM) versus intensity has an formula of Intensity = 509067 · concentration + 792496

Figure 6. Calibration curve of CsgA concentration (µM) versus intensity.

We calculated from figure 6 that the average intensity of our samples was 2.3106 Au. We calculated, using the calibration line, that the concentration of the internal CsgA_His proteins was equal to 2.8μM. The amount of sample we loaded on the gel was 10 µl, therefore the amount of CsgA_His protein in the lane 2.75·10^-5µmol.

Because in the beginning we had an OD600 of 10, it means that we had a concentration of 10·8·108 cells/ml. Therefore, the CsgA_His came out of this amount of 8·10⁷ cells in 10µl. By dividing the amount of CsgA_His protein by the amount of cells, we obtained an internal CsgA_His protein amount of2.82·10^-7 µmol/cell, or 2.82·10^-13 mol/cell. With the constant of Avogadro 6·10²³ molecules/mol, we calculated an end internal CsgA_His protein amount is equal to 2.06·10⁵ molecules/cell.

Transmission electron microscopy

Using TEM the formation of curli of the biobrick BBa_K1583100 was visualized. Although this is a different biobrick containing only CsgA (compared to the fusion protein of Mfp5_CsgA_His), curli formation can be presumed to be similar for this biobrick suggested by the Crystal Violet assay.

Fig. 5: TEM images (magnification 7300 x) of cells containing BBa_K1583100. The left picture shows uninduced cells (0% Rhamnose).The picture on the right shows cells incubated with 1% (w/v) rhamnose.

We did not observe formation of curli nanowires in the uninduced cultures of our strain. However, cells from induced cells clearly produced them, as supported by the TEM images.

Reference

Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”,

Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2

Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66

Zhou, Kang, Kangjian Qiao, Steven Edgar, and Gregory Stephanopoulos. 2015. “Distributing a Metabolic Pathway among a Microbial Consortium Enhances Production of Natural Products.” Nature Biotechnology 33(4): 377–83.