Part:BBa_K1583108

pRha + CsgA with Hydroxyapatite-affinity tag & pTet + RFP

CsgA with Hydroxyapatite-tag attachted to the C-terminus under control of L-rhamnose-inducible promoter (BBa_K914003). Addionally a RFP coding device was added (BBa_I13521). The hydroxyapatite-affinity tag generates a peptide tag which shows high adhesive properties towards hydroxyapatite, a main component of e.g. teeth. The sequence of the hydroxyapatite-binding peptide is from an article by Roy et al. 2008.

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. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG.

In order to improve BBa_K1583102 we added BBa_I13521 constitutively expressing RFP. This could be used for both screening for successful transformations, as for visualisation of cells with fluorescent imaging.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 635
  Illegal AgeI site found at 747
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Four different experiments were done to characterise the BBa_K1583102 biobrick:

Fluorescence assay (Rhamnose inducible expression)

Crystal Violet assay (Curli formation)

Affinity assay using Bos taurus dental parts (Hydroxyapatite affinity)

Transmission electron microscopy (Curli formation)

As shown in the crystal violet assay in the characterization section of pRha+CsgA & pTet+RFP ((BBa_K1583106)), the RFP conding device does not affect the formation of CsgA protein. Therefore the characterization data of BBa_K1583102 is also valid for this biobrick.

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 CsgA with a Hydroxyapatite-affinity 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_HA-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.).

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. HA0, HA2 and HA5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains CsgA attached to the hydroxyapatite tag 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).

The CsgA_HA proteins seem to have retained the capability to form curli despite having a peptidic modification on the C-terminus. 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 & HA2	0.0015	Yes
CTRL5 & HA5	0.0152	Yes

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

Affinity assay using Bos taurus dental parts

In our research for a highly representative biofilm that could be used for testing products, we thought about the main surfaces where a non-desirable biofilm can be attached. The first structure that came into our minds was the dental cavity; there, biofilms can attach to the tooth cover and create strong and resistant biofilms (Kidd, E.A., et al, 2004).

Figure 3. Teeth from a cow (Bos taurus) used in the experiment for testing the hydroxyapatite peptide tag

For the experiment aiming to prove that our biofilm can be strongly linked to the enamel surface, we managed to get some teeth from a cow (Bos taurus) (Figure 3.). We cut them in small pieces of similar size (~1cm3) and place them in a 96-well plate. Afterwards the well was filled with 5 mL of cell culture, induced and incubated for 40 hours at room temperature, without shaking. The strains used for the investigation are summarized in table 1:

Table 1: Summary table of the strains used for the experiment of hydroxyapatite attachment.

Strain	Plasmid	Gene	Function in the experiment
E. coli K-12 MG1655 PRO ΔcsgA ompR234	BBa_K1583102	CsgA+HA Tag	Test
E. coli K-12 MG1655 PRO ΔcsgA ompR234	BBa_K1583100	CsgA	Test
E. coli K-12 MG1655 PRO ΔcsgA ompR234	pSB1C3	Empty plasmid	Control

Once the dental pieces had enough time to be covered with specific curli-generating cells, we designed two different protocols for measuring the efficiency of the affinity tag. In both cases, the cells were expressing RFP (red fluorescent protein) under a constitutive promoter. Thus, the number of cells that remained attached to the surface could be calculated from the fluorescence intensity.

We tried to measure the remaining cells attached to the dental surface after submerging it in water for 2 seconds. The first approach included a direct measurement technique: measuring RFP presence using a Typhoon ® Fluorescence Scanner.

On the other hand, we also measured the cells attached to the tooth by putting the samples in an ethanol (70%) solution for 5 minutes. Consequently, the cells detached and float in the supernatant. When we measure the fluorescence intensity of this ethanol supernatant, we were able to discriminate if there is actually a difference in the attachment properties of tagged and untagged cells. Detailed information of the nomenclature used in the figures of this section can be found in Table 2.

Table 2: Summary of the samples analysed after the cells grew on them.

Sample	Gene	Rhamnose	Function in the experiment
Ha+	CsgA+HA Tag	Yes	Test
HA-	CsgA+HA Tag	No	Control
CSGA+	CsgA	Yes	Test
CSGA-	CsgA	No	Control
PSB+	Empty plasmid pSB1C3	Yes	Control
PSB-	Empty plasmid pSB1C3	No	Control

We tested the RFP presence in the tooth samples (figure 4) with a fluorescence scanner, obtaining the following results (figure 5):

Figure 4: Dental samples used in the experiment of hydroxyapatite affinity. From left to right, the samples are HA+, HA-, CSGA+, CSGA-, PSB+, PSB-. All the tooth fractions are approximately of 1 cm3.

Figure 5: Fluorescence scanning of the teeth samples with an excitation wavelength of 532 nm and emission detection at 610 nm. The samples analysed were HA+ (top left), HA- (top centre), CSGA+ (top right), CSGA- (bottom left), PSB+ (bottom centre) and PSB- (bottom right).

The results of this experiment showed a strong background from the samples, meaning that probably the teeth display some kind of self-fluorescence. Consequently, the results of this test cannot be considered for proving the affinity to hydroxyapatite of our biofilms.

Then we performed the second measurement assay, using a 96-well plate and the plate reader. The results are displayed in figure 6, and the parameters were confirmed using the supernatant of the CsgA producing cells with no ethanol treatment.

Figure 6: Fluorescence read of the ethanol supernatant where the dental samples where incubated. The liquids were excited with 560 nm of wavelength and the emission length detected was 607, for RFP fluorescence detection. All the samples are measured in triplicates, and the error bars show their standard deviation.

From the results shown above, we could conclude that our affinity tag for hydroxyapatite gives an actual difference when compared with the control samples, for significance level of 5%. Also, the statistical analysis confirms that there is a significant change between cells with the affinity tag and without it (Table 3).

Table 3: Significance analysis of the samples analysed. All the samples but the empty plasmid control display a significant difference when compared to the blank. Thus, both CsgA and CsgA+HA tagged cells attach better to a dental surface than cells that do not express the amyloid curli. Furthermore, the difference of fluorescence between the cells expressing the tagged protein and the untagged is also significantly different, confirming the efficiency of the hydroxyapatite peptide tag.

Samples compared	p-value	Significatant difference (5%)
Ha+ % ETOH	0.0014	Yes
CSGA+ & ETOH	0.0202	Yes
PSB+ & ETOH	0.7295	No
CSGA+ & HA+	0.0008	Yes

Transmission electron microscopy

Using TEM the formation of curli of the biobrick BBa_K1583100 was visualized. Although this is a different biobrick (no addition HA-tag), curli formation can be presumed to be similar for this biobrick (Figure 7).

Figure 7: 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

Roy, M. D., Stanley, S. K., Amis, E. J., & Becker, M. L. (2008). Identification of a Highly Specific Hydroxyapatite-binding Peptide using Phage Display. Advanced Materials, 20(10), 1830–1836. p>

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.