Problem and Solution

Biofilms are communities of bacteria connected by protein nanowires and surrounded by an extracellular matrix. In this form, they are more resistant and can severely affect human health, industrial productivity and the environment. More precisely, biofilms can cause infections in the human body, affect water quality, and damage industrial installations and equipment. Research and industry have been working to find various solutions for preventing and removing this threat. Potential solutions, such as health products, drugs and industrial removal products, are tested on artificially formed biofilms.

The problem with biofilms formed artificially is that they are time consuming, difficult to control and to reproduce. This means that artificial biofilms do not reflect natural biofilm characteristics, making product testing unreliable. Therefore, biofilm-removal products may have a different effect when used in natural settings, with unforeseen negative side-effects and reduced efficiency.

Our project is entitled Biolink and provides an alternative to current biofilm formation technologies. We use a 3D printer, which we call The Biolinker, to form layers of a designed bioink made of bacteria that can bind together into a desired structure.

Biolink helps biofilm-related industries in several ways. First, it brings reproducibility and control to how bacterial biofilms can be artificially formed. Second, biofilm printing adds automation and scalability, making biofilm formation processes more efficient, and thus, cheaper. Hence, Biolink can help to design safer and more effective anti-biofilm solutions by increasing biofilm testing process efficiency and resemblance to reality.

The Biolink Project

Our printing system, called Biolink, can be summarized in the following sentence: biofilm producing bacteria are printed with the help of a flexible scaffold hydrogel. First of all, our homemade bacteria (modified to make biofilms) are mixed with a solution of sodium alginate and subsequently with calcium chloride. There, the Ca2+ molecules keep the structure fixed creating a stable gel. This hydrogel-bacteria mixture is then induced with rhamnose, a sugar specific for our promoter, which makes them synthesize CsgA, the linking molecule. CsgA proteins polymerize to an amyloid structure surrounding the cells and connecting them to each other through the scaffold. Once the cells are all attached in the structure defined by the gel scaffold, it is no longer necessary. Consequently, the hydrogel is dissolved with sodium citrate. But the cells are still connected due to the curli amyloid! So, we obtain a perfectly defined 3D structure made of bacteria.

The Biolink project promotes the open source and educational spirit of iGEM. Our 3D printer, the Biolinker, is made of K’NEX construction toys, a DIY solution that is both easy to build and efficient in doing its job. Through policy and practice we try to position our project within the synthetic biology industry and academia, as well as observe socio-economic perception and feedback. We accomplish this by analyzing and interviewing stakeholders, treating ethical and regulatory issues, and building a business plan around our project.

Synthetic Biology in our Project

During biofilm formation, bacteria produce an extracellular matrix made of amyloid structures. These amyloid structures are curli fimbriae, composed of intertwined filaments with a thickness of approximately 4-7 nm (Nguyen, 2014). Therefore, curli production helps bacteria bind to each other in natural biofilms (Taylor et. al. 2012). There are two distinctive operons involved in this highly regulated pathway; csgBA and csgDEFG. The csgBA operon encodes for two proteins: CsgA and CsgB. The csgDEFG operon encodes for the proteins required for the transport of CsgA and CsgB to the cell surface (Dueholm et al, 2011).

On one hand, CsgA is an amyloid protein that acts as monomer for curli formation. On the other hand, the protein CsgB in an integral membrane protein, which binds CsgA to the cell; CsgB acts as an anchor for curli formation. CsgA is transported as an unfolded protein to the extracellular matrix. Once outside the cell, it aggregates with CsgB and the self-assembly of these aggregates form the amyloid fibrils. When CsgA comes in contact with CsgB, the fibrils bind to another cell and the process is repeated again until an entire network has been created (Barnhart et al, 2006).

In our project, we designed an inducible system for synthesizing CsgA in biofilm-making deficient cells. Besides to that, we aimed to create a customized biofilm. To do so, we designed different biobricks that contain a peptide tail attached to the sequence of the biofilm protein CsgA that provides a specific surface affinity. In the end, we planned to use our engineered cells (which also express a fluorescent reporter) for printing in different layers; printing biofilms in 3 dimensions.

The Bioink and Alginate as Supporting Scaffold

In initial experiments printing with bacterial cells dispersed in LB media it quickly became obvious, that a supporting scaffold would be required: due to surface interactions the liquid spread out creating a final thickness of the printed line of almost one centimeter. In tissue engineering sodium alginate is commonly used as a synthetic extracellular matrix material (Rowley et al., 1999). Inspired by this, we took a look at sodium alginate to use as a scaffold material. Sodium alginate is a carbohydrate polymer which can be fairly well dissolved in water, but in contact with calcium ions (or other divalent cations) the polymers are connected via electrostatic interactions forming a hydrogel. Made for instance from LB, this hydrogel could provide bacterial cells with everything they need for weeks and keep them alive. Furthermore, jellification is a reversible process by complexing the calcium ions with citrate and replacing it again with sodium ions (Rowley et al., 1999). Thus, we had found a substance ideally meeting our purpose of being initially liquid, capable of rapidly turning into a viscous gel and reversing this process again.

Escherichia coli K-12 MG1655 PRO ΔcsgA ompR234

This strain of Escherichia coli, used in previous studies of amyloid fiber production in bacteria, is characteristic for having knocked-out the CsgA gene. However, it has all the other genes required for formation of the curli structure.

We have used this strain for our project as the main organism for the printing process. As the bacteria cannot express csgA, we transformed our strains with a plasmid containing this gene under the control of an inducible promoter. Consequently, we can modulate where and when the amyloid fiber will be formed! (Chen, A.Y., et al. 2014)

Escherichia coli Top10

This strain was used exclusively for highly efficient transformations. We used this organism for cloning experiments, plasmid isolations and other basic steps of our project.

Introduction and Motivation

Printing a biofilm requires the bacteria to produce several components that allow them to make a connection between them. Furthermore, they require to attach to the surface where they are printed on. Finally, they need to make the biofilm only when it is desired.

In order to study the connections that exist between bacteria, we focused on the bacterial amyloid curli structure. These bacterial structures contribute to biofilm performances where cells interact with other cells and even surfaces. The curli consists of proteins bound together and to the cell membrane. CsgA, the main subunit, polymerizes in the extracellular space creating an amyloid nanowire; and CsgB anchors this nanowire to the membrane, creating connections between different cells (Joshi et al., 2014).

Consequently, we designed different versions of biobricks (see Parts) coding for the CsgA gene to create a biofilm in a controlled manner from CsgA deficient cells. To prevent premature biofilm formation and clogging of our printer, we designed the expression of CsgA to be inducible by rhamnose so that the biofilm is only created after the cells are printed. This also functions as a safety feature in case of escape. The experiments (and their results) that we used to analyse the expression of CsgA and its promoter are explained in this section.

Fluorescence assay for kinetic measurements regarding induction of csgA expression

Background

In the kinetic experiment, the fluorescence signal of the proteins CsgA and CsgA-GFPmut3 (I13504) proteins expressed by our constructs was recorded in time. Besides fluorescence, we measured the OD600 of our cultures in order to normalize the fluorescence signal per cell. The main goal of this experiment was to see, if different induction levels with increasing rhamnose concentrations would lead to a higher production of CsgA. If this was true, the quantity of produced CsgA present in the medium could be controlled. This degree of control is key to achieving our main goal; making a biofilm with reproducible strength.

The obtained results can be used to calculate the promoter strength at different induction levels of rhamnose in our model. In order to use the mathematical model previously constructed in the modeling section, it has to be fitted to GFPmut3 units/cell/second. Therefore, we set up a calibration curve with exactly the same settings as the fluorescent experiment in order to correlate fluorescence signal to units of GFPmut3 per cell.

Methodology

In this experiment, the strains ∆csgA – csgA- I13504 and ∆csgA – csgA strain were both induced with rhamnose at different concentrations. The different induction levels can be found in Table 1.

All conditions (ID 1 – 6) were carried out in triplicates for accurate statistical analysis of the data. The different cultures were grown and induced in a 96-well plate. The OD600 and the fluorescence signal was recorded with a plate reader during an 18-hour period at 30°C.

Results and discussion

In Figure 5, the fluorescent signal was normalized with the number of cells and plotted as a function of time. The red bars denote the error within each ID.

As observed in Figure 5, only the strains carrying the csgA-GFPmut3 construct induced with 0.2% (w/v) and 0.5% (w/v) showed a clear increase in fluorescence signal over time. The rest of the cultures, didn’t show significant fluorescence over time.

Furthermore, we have showed that increasing concentrations of rhamnose lead to increasing amounts of produced csgA-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 directly after induction, but activity is observed after a time period of 3 to 4 hours. This is in accordance with data from literature (Wegerer et. Al, 2008), in which low fluorescence levels were observed after 2 hours of induction of the rhamnose promoter.

The calibration line of fluorescence versus mass amount GFPmut3 is given in Figure 6.

The corresponding function of the GFPmut3 calibration line is:

With mass_GFP in ng.

In the modelling, the fluorescent data in Figure 5 will be further converted to molecules GFPmut3/cell and the promoter activity will be calculated for both the 0.2% (w/v) and 0.5% (w/v) level of rhamnose induction. With this kinetic experiment, we have proven that our csgA-GFPmut3 construct is able to produce different levels of GFPmut3 by varying the rhamnose concentration.

Transmission Electron Microscopy (TEM) for visualization of bacterial amyloid curli

Microtiter Plate Assay for Ring-Shaped, curli dependent biofilm detection

Quantification of the internal CsgA concentration using Western Blot

Conclusions and Future Directions

Introduction and Motivation

Conclusions and Future Directions

Introduction

Conclusions and Future Directions