Difference between revisions of "Part:BBa K1583101"

Revision as of 09:24, 18 September 2015

pRha + CsgA + His-tag

CsgA with HIS-tag attachted to the C-terminus under control of L-rhamnose-inducible promoter ((<a href="https://parts.igem.org/Part:BBa_K914003">BBa_K914003</a).

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.

Characterization

Four different experiments were done to characterise the BBa_K1583101 biobrick:

Fluorescence assay

To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (<a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112">BBa_K1583112</a>) 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.

<figure> <img src="" width="100%" height="100%"> <figcaption> 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. </figcaption> </figure>

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> <img src="" width="70%" height="100%"> <figcaption> Fig. 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. HIST0, HIST2 and HIST5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains csgA attached to a 6xHis 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). </figcaption> </figure>

The CsgA_His 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). </html>

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.

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.

Sequence and Features

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

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.