PROTEORHODOPSIN

Our goal is to remove as many NPs as possible from wastewater systems to minimize damage to the environment and our health. Due to the sheer variety of manufactured NPs and the significant chemical differences between them, we looked for common properties across different types of NPs. Most industrially produced NPs are coated with capping agents to prevent aggregation (ref). We specifically target citrate, the most common NP capping agent used in consumer products (Levard et al. 2012).

Construct Design

PR is a transmembrane protein found in proteobacteria. Since PR has previously been shown to bind citrate through two positively charged lysine residues on its surface (figure 2-1; Béjà et al. 2000; Syed 2011), we hypothesized that PR may also bind citrate capping agents and CC-NPs.

We obtained the DNA sequence of pR (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). The sequence of pR was then flanked by an upstream strong promoter and strong ribosome combination (BBa_K880005), and a downstream double terminator (BBa_B0015) to maximize expression of PR protein. This final construct (BBa_K2229400; figure 2-2) was ordered from IDT and cloned into pSB1C3, a biobrick backbone.

Functional Test: Does it trap CC-NPs?

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Characterization: How many NPs can we trap?

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BIOFILM

Our biofilm approach was originally inspired by the ability of jellyfish mucus to bioaccumulate gold NPs (AuNPs) and quantum dots through electrostatic interactions (Patwa et al. 2015). A biofilm is a community of microbes embedded in a matrix of extracellular polymeric substances (EPS) consisting of different polysaccharides, proteins, and lipids (Donlan 2002). Recent studies show that the EPS in biofilms can also trap various NPs (Kaoru et al. 2015, Nevius et al. 2012) (figure 3-1).

Most wastewater treatment plants (WWTPs) already use microbes to break down and remove organic components in wastewater (Sehar & Naz 2016). The recent increase in NP contaminants entering WWTPs, however, poses a threat to these microbes and the treatment process. For example, silver NPs (AgNPs) have antimicrobial effects and other metal oxide NPs can inhibit microbes from performing important processes such as nitrification to remove ammonia (Yun & Lee 2017; Walden & Zhang 2016). Biofilms are nearly four times more resistant to NPs compared to planktonic bacteria, increasing their tolerance to NPs in wastewater (Choi et al. 2010).

Can Biofilms Trap Nanoparticles?

The first step of this approach is to test if an E. coli biofilm can effectively trap NPs. E. coli liquid cultures grown in Luria-Bertani (LB) broth were transferred to petri dishes, each containing glass coverslips to provide a surface for adherence. The dishes were incubated at 37˚C for 7 to 14 days, with 2 mL of LB added every two days to prevent the media from drying out. Viscous, film-like substances began to develop, indicating the production of EPS and biofilm (figure 3-2). At this point, we could extract and wash the biofilm.

Hoping to image the structure of biofilms up close using scanning electron microscopy (SEM), we tried different fixation and preparation methods (figure 3-3 & table 1) (Fischer et al. 2012): 1) freeze drying with a critical point dryer (CPD), 2) fixation with glutaraldehyde (GA), and 3) fixation with a combination of Alcian Blue, GA, and K₄Fe(CN)₆. We further improved image quality by taking multiple images of a field of view and stacking the images together to reduce noise (figure 3-4).

To test if biofilms can trap NPs, we used a solution containing 30 nm AuNPs (from Sigma Aldrich). Because the AuNP solution is purple in color, we can take absorbance measurements and convert these values to AuNP concentration via Beer’s Law. Four experimental groups were set up (figure 3-5): a negative control containing only AuNPs, and three tubes containing AuNP with either planktonic bacteria, biofilm, or “dead” biofilm (treated with antibiotics). The four groups were shaken for 24 hours and centrifuged to isolate the supernatant (figure 3-5, C). The supernatant contains free AuNPs, which were measured using a UV-VIS spectrometer at 527 nm.

The negative control and the AuNP with planktonic bacteria groups did not change the purple color of the AuNP solution, indicating that bacteria alone cannot trap NPs (figure 3-5, C and D). The addition of biofilm, however, greatly reduced the amount of AuNP in the supernatant. Even with antibiotics, AuNP levels in the supernatant were still reduced, suggesting that the removal of AuNPs depends on the sticky and slimy extracellular components of biofilm and not on the bacteria itself. When we fixed and imaged the biofilm + AuNP sample by SEM (figure 3-6), we could see NPs in EPS areas. This confirmed our idea that NPs were being trapped in the EPS layer of biofilms.

How Is Biofilm Production Regulated?

In E. coli, biofilm synthesis is mainly mediated through two curli operons (Barnhart & Chapman 2006). Curli fibers facilitate cell-surface and cell-cell adhesion, biofilm synthesis, and are the main protein components of the EPS (Reichhardt et al. 2015, Barnhart & Chapman 2006). The operons (csgBA and csgDEFG) control the expression of six proteins essential to biofilm formation (figure 3-7). CsgA and CsgB are curli monomers which can polymerise to form curli fibers. CsgD is an activator of csgBA transcription (figure 3-7). CsgE, CsgF, and CsgG help export CsgA and CsgB out of the cell. The operon csgDEFG can be activated by the protein OmpR, and the subsequent expression of all six proteins increase biofilm formation (Barnhart & Chapman 2006).

Construct Design

Three constructs were built to upregulate curli production by overexpressing CsgD, OmpR234 (a mutant form of OmpR which is constitutively active), or both. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), csgD (BBa_K805015), ompR234 (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.

For strong CsgD expression (figure 3-8, BBa_K2229100), csgD was inserted behind BBa_K880005 (figure 3-10, BBa_S05397), and then before BBa_B0015 (figure 3-11).

For strong OmpR234 expression (figure 3-9, BBa_K2229200), ompR234 was inserted before BBa_B0015 (figure 3-10, BBa_S05398) and then behind BBa_K880005 (figure 3-11).