Coding

Part:BBa_K1583002

Designed by: Stefan Robert Marsden   Group: iGEM15_TU_Delft   (2015-09-04)

Mfp5 adhesive protein

This basic part contains the Mfp5 gene. It is used in the biobricks BBa_K1583104 and BBa_K1583110 as a fusion protein with the protein CsgA (BBa_K1583000).The expression of this fusion protein can be controlled with the rhamnose inducible promoter (BBa_K914003).

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. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. By creating a fusion protein of Mfp5 and CsgA, 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).

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 76
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

This basic part was used in the biobricks BBa_K1583104 and BBa_K1583110. These biobricks were characterized in three different experiments:

  • Protein expression
  • Fluorescence assay
  • Crystal Violet assay
  • 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 figure 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.

Figure 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 the fusion protein of Mfp5 and CsgA 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 Mfp5_CsgA-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 2.).

Generic placeholder image
Figure 2: 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. MFP50, MFP52 and MFP55 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains the Mfp5_CsgA fusion protein under an rhamnose 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).

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 1.).


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

    Sample p-value Significant difference (5%)
    CTRL2 & MFP52 0.0043 Yes
    CTRL5 & MFP55 0.0016 Yes


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).


Transmission electron microscopy

Using TEM the formation of curli of the biobrick BBa_K1583100 was visualized (Figure 3). 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.

Figure 3: 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

Wegerer, A., Sun, T., and Altenbuchner, J. (2008). Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, BMC Biotechnology, 8:2

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.

Improved by team Greatbay_SCIE

Characterisation

BBa_K1583002 was characterized in following experiments:

  • Protein expression
  • Protein purification

Protein expression

Figure 4. The circuit of the protein, with a His-tag.

In our project, Mfp5 was used as an adhesive part to create recombinant proteins. We clone Mfp5 alone into the pET28b expression vector as control. We expressed pET28b-mfp5 in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no obvious protein bands of Mfp5 (~10 kDa) could be observed on lane mfp5 compared with lane pET28b (pET28b empty vector)(Figure 5A), which means the expression of this protein couldn’t be detected in BL21(DE3) Rosetta. Quantitative densitometry analysis of SDS-PAGE indicated that Mfp5 almost not expressed under this expression conditions (Figure 5B).

Figure 5. Detection of the expression level of all recombinant proteins by SDS-PAGE. (A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots.

Protein purification

The predicted size of mfp5 is 9.97 kDa and the isoelectric point is 10.19. Though no bands of interest could be observed on gels detecting the expression of Mfp5 (Figure 5A), we straightly go protein purification under denaturing conditions. As shown in Figure 6, no bands around 10kDa could be observed on Lane E1 and E3, which were eluted protein samples.

Figure 6. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein Mfp5 by cobalt-resin columns. Lanes: M, protein molecular weight marker; WC, whole-cell sample of recombinant proteins; IS, insoluble fragments. E, eluted proteins. 12% SDS-PAGE gels were used for the analyses.

[edit]
Categories
Parameters
None