Team:TAS Taipei/Experimental Summary
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Project
Experiment
Modeling
Prototype
Human Practice
Biosafety
About Us
Attributions
Experiments
Constructing DNA to make our ideas come to life.
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EXPERIMENTAL SUMMARY
We use two approaches to capture NPs: 1) target citrate-capped nanoparticles (CC-NPs) using a bacterial membrane protein Proteorhodopsin (PR), and 2) trap a wide variety of NPs using an E. coli biofilm. [pR outline/conclusion] We show experimentally that an E. coli biofilm can trap gold and silver NPs ranging from 15-60 nm. To increase biofilm production, we overexpress CsgD and OmpR234, two positive regulators of biofilm synthesis. We demonstrate that E. coli carrying our constructs produce up to 8 times more biofilm compared to controls.
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.
Figure 2-1. PR-citrate interaction (Syed 2011). Two lysine residues (blue) on the surface of PR can bind citrate (red).
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.
Figure 2-2 Construct expressing pR. Our construct includes a strong promoter, strong RBS, pR and double terminator.
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).
Figure 3-1. We envision using E. coli biofilms (green) to trap nanoparticles of different sizes and composition (pink).
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.
Figure 3-2
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).
Table 1
Figure 3-3
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.
Figure 3-5
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.
Figure 3-6 SEM Image showing AuNPs trapped by biofilm. A biofilm+AuNP sample was fixed with GA. Some EPS is preserved (red) and AuNPs (white) seemed to aggregate and adhere onto the EPS.
How Is Biofilm Production Regulated?
Figure 3-7 Two curli operons—csgBA and csgDEFG—direct biofilm synthesis.
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.
Figure 3-8 CsgD expression. Our construct includes a strong promoter, strong RBS, csgD and double terminator.
Figure 3-9 OmpR234 Expression Our construct includes a strong promoter, strong RBS, ompR234 and double terminator.
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).
Figure 3-10 PCR Check for BBa_K880005+CsgD and OmpR234+BBa_B0015. The expected size of BBa_K880005+CsgD is 1000 bp (orange box) and OmpR234+BBa_B0015 is 1100 bp (blue box).
Figure 3-11 PCR Check for CsgD, OmpR full constructs. The expected size of CsgD full construct is 1100 bp (orange box) and OmpR full construct is 1200 bp (blue box).
Figure 3-12 CsgD and OmpR234 Expression Our construct includes a strong promoter, two strong RBS, csgD, ompR234 and double terminator.
To strongly express both CsgD and OmpR234 (figure 3-12, BBa_K2229300), a strong RBS (BBa_B0034) was inserted in front of the intermediate BBa_S05398 (ompR234+double terminator) to make BBa_S05399 (RBS+ompR234+double terminator). FInally, BBa_S05397 (K880005+csgD) was inserted before BBa_S05399 to complete the full construct (BBa_K2229300) (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.
Figure 3-13 PCR Check for BBa_K2229300. The expected size of BBa_K2229300 is 1900 bp (green box)
Hypothesis
Figure 3-14 Overexpression of CsgD and/or OmpR234 upregulates the curli operon to different different degrees We hypothesize that biofilm production would be upregulated (in increasing order) if we overexpress A) CsgD, B) OmpR234, or C) both.
We hypothesized that biofilm production would be upregulated (in increasing order) if we overexpress CsgD, OmpR234, or both (figure 3-14). Overexpression of CsgD would result in more curli monomers, but no transport proteins to carry the monomers out of the cell. Overexpression of OmpR234 would allow curli monomers to be exported and form fibers and biofilm. Finally, when both CsgD and OmpR234 are overexpressed, twice the amount of curli monomers should be made and exported to form fibers and biofilm.
Expression of CsgD and OmpR234 Increases Biofilm Formation
To test the expression of CsgD and OmpR234, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). Cultures transformed with the basic parts BBa_K805015 (csgD ORF alone) and BBa_K342003 (ompR234 ORF alone) were used as negative controls. We expected to see CsgD around 25 kDa and OmpR234 around 27 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to negative controls, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) and BBa_K2229200 (OmpR234 overexpression) (figure 3-15; proteins bands are marked by asterisks).
In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012) . This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in figure 3-15).
Figure 3-15
After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips, and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-16, 3-17, 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
We find that overexpressing CsgD and/or OmpR234 increases biofilm production to different degrees, as we hypothesized (figure 3-19). Overexpression of CsgD (BBa_K2229100) doubles biofilm production compared to the negative control BBa_K805015 (figure 3-16); overexpression of OmpR234 (BBa_K2229200) leads to about 8 times more biofilm compared to the negative control BBa_K342003 (figure 3-17 & figure 3-18, A). CGU_Taiwan helped us independently verify our OmpR234 overexpression results using a different dye, crystal violet, which is also commonly used to stain biofilms (figure 3-18, B). Interestingly, both biofilms characterized in our assay are found around the glass coverslip and do not seem to stick well to the glass surface (figures 3-16 & 3-17).
Figure 3-16
Figure 3-17
Figure 3-18
When all three constructs were compared, we find that overexpression of both OmpR234 and CsgD (BBa_K2229300) increased biofilm production the most (figure 3-19). BBa_K2229300 also increased adhesion to our glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps (figure 3-19, A).
Figure 3-19
In summary, we demonstrate that biofilms can trap NPs and our constructs function as hypothesized. Our collection of constructs (BBa_K2229100, BBa_K2229200, and BBa_K2229300) can successfully upregulate biofilm production to varying degrees.
*Details on any experimental setup can be found in the Protocols section of our lab notebook.
REFERENCES
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