Part:BBa_K1583106

pRha + CsgA & pTet + RFP

This part was designed to express the csgA gene under control of L-rhamnose-inducible promoter. Addionally a RFP coding device was added (BBa_I13521).

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.

Visualisation is the key when it comes to analysing your samples and drawing conclusions. In order to improve this, we added BBa_I13521 constitutively expressing RFP to our device giving our cells a nice, red fluorescent glow. We used this as a quality check to ensure that the cells we were working with still contained our specific plasmid DNA.

This part was designed to be able to control the nanowire formation.

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

This part was characterized in three different experiments:

• Fluorescence assay
• Crystal Violet assay
• Transmission electron microscopy

As shown in the crystal violet assay in the characterization section of pRha+CsgA & pTet+RFP (BBa_K1583106), the RFP coding device does not affect the formation of CsgA protein. Therefore the characterization data of BBa_K1583101 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 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 CsgA-producing bacteria can 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-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 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.).

Fig. 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. CSGARFP0, CSGARFP2 and CSGARFP5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains csgA under an inducible promoter and RFP under a constitutive promoter. The termination "0", "2" and "5" denotes the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).

This shows that this biobrick does not affect the CsgA production compared to the biobrick that only contains the csgA gene (BBa_K1583101). 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. 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 when comparing to the empty plasmid, with a significance α value of 5%.

Sample	p-value	Significant difference (5%)
CTRL2 & CSGARFP2	0.0009	Yes
CTRL5 & CSGARFP5	0,0503	No

With the experiments described previously we achieved nanowire formation with strains containing the BBa_K1583100 biobrick.

Transmission electron microscopy

In order to determine the persistence lenght of the nanowires, we wanted to visualize the nanowires produced by our engineered bacteria. To be able to do so we used TEM to image the cells (Figure 3).

Fig. 3: TEM images (magnification 7300 x). 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.