Part:BBa_K5177000

GsPilA-TKKTLRT

This part encodes the E.coli codon optimised GspilA monomer with a mammalian collagenase-derived collagen I binding peptide tag. pilA includes an upstream E.coli ppdD signal peptide for Geobacter sulfurreducens pili expression in E.coli DH5α.

The TKKTLRT collagen binding heptapeptide was first deduced from the region in human α2(I) collagen surrounding the bond between Gly⁷⁷⁵ and Ile⁷⁷⁶ using the approach of sense–antisense peptide interactions. A polyclonal serum against this peptide can recognize purified human collagenase. Collagen can bind specifically and quantitatively to this peptide in a saturable fashion.[1] The Kd (Binding Affinity) of the TKKTLRT peptide is ≤100μM.[2]

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Structure and Conductivity

The pili of Geobacter sulfurreducens are filaments primarily made up of the PilA protein monomer. Due to the short length of the monomer, aromatic residues (tryptophan, phenylalanine, and tyrosine) can be tightly packed along the pili’s length, providing it with conductive properties [3]. This characteristic can be explained by the phenomenon of pi stacking, in which the aromatic side chains align, creating a path for electron transfer to occur [4]. The relationship between the PilA's conductive properties and its protein sequence has further been proven by genetic engineering. For instance, conductivity of pili in which the five aromatic amino acids of the monomers are substituted with alanine is shown to drop to 38 ± 1 μS cm−1, three orders of magnitude lower than the native pili, underscoring the significance of the pi interactions in electron transfer [5]. Furthermore, pH also impacts the conductivity of pili; ranging from 51 mS/cm3 at pH 7 to 37 mS/cm3 at pH 10.5, with the highest conductivity value, 188 mS/cm3, observed at pH 2 [5]. Consistent with this, purified pili films demonstrate non-redox electronic conduction in a buffered aqueous environment; pili can conduct electrons without going through redox processes, which is in line with a metallic charge carrier transport model. This comes in sharp contrast to other organic reactions (e.g. DNA synthesis, photosynthesis), which follow a donor–bridge–acceptor model, where electric potential is passed on by the transfer of electrons from one molecule to the next until it reaches the final acceptor.This also explains how the conductivity of the pili is comparable to synthetic conductive materials, such as carbon nanotubes or silicon nanowires [6] [7].

Potential Applications

Over the last decade, research on pili has revealed a number of potential applications for G. sulfurreducens. For instance, microbial fuel cells can be constructed by connecting a G. sulfurreducens biofilm with the organic waste (e.g. acetate), generating electricity through pili-mediated reduction, [8]. Similarly, the capacity of natural Geobacter to reduce and detoxify radioactive elements and heavy metals in contaminated environments through pili-mediated reductions (e.g soluble Uranium VI to Uranium IV, a form that precipitates and is distributed at a lower rate) can also be enhanced, creating more efficient systems for clearing radiologically and chemically contaminated areas [9] [10]. Electrical biosensors, which could have medical applications in the future for the detection of relevant metabolic compounds, can be created by modifying the C-terminal end of the pilin monomers. Hybrid pili, which consist of wild type, as well as his-tagged and HA-tagged monomers have been shown to conduct electricity better than the wild type pili, as well as change their conductivity potential when binding to the target molecule nickel [11] [12]. Finally, G. sulfurreducens can serve as a model organism for microbially-induced corrosion in metal structures, such as storage tanks and pipes; understanding the electrochemical mechanism of this process can pave the way for technologies that lower maintenance requirements and increase the longevity of these buildings [13].

Allergenicity

The part was evaluated for allergenicity through comparison with identified allergenic protein sequences present in the University of Nebraska Lincoln allergen sequence database (http://www.allergenonline.org) according to a protocol from the 2017 iGEM Baltimore Bio Crew (https://2017.igem.org/Team:Baltimore_Bio-Crew/Experiments).

In full-length alignments, % identity of 50% and above means the part is a potential allergen. Additionally, in an 80 amino acid sliding window, a similarity lower than 35% further reduces the probability of the part being an allergen. For BBa_K5177000 the value was 31.7% identity with the top allergens in the database for both cases, indicating it is unlikely to be of potential allergen status.

Experimental Results

Dry Lab

Our dry lab results for AlphaFold modelling of the monomer with the collagen tag suggest that the monomer properly folds even after addition of a tag, and as prior work has yielded tags of 9 amino acids in length (Ueki et al. 2019) we decided to try and express the collagen-binding protein nanowire.

Figure 1. Structure of GsPilA monomer with TKKTLRT Collagen I Binding Tag (In orange)

We also analysed a homology model of the Geobacter sulfurreducens assembled pilus which suggests the tags on the C-terminus of GsPilA are expressed on the outer surface of the e-pili.

Figure 2. From Malvankar et al. (2015) Homology Model of G. sulfurreducens pilus constructed with a Pseudomonas aeruginosa template. This suggests amino acids on the C-terminal end are exposed on the outer surface of the e-pili. [11][14]

Consequently, we proceeded to do our wet lab protein expression and binding assay.

Binding Assay

DH5α ΔsfmA cells transformed with RFP (S1a: vector pBbS1a-RFP, Addgene ID: JPUB_000103 ), type IV assembly machinery (A1k: pBbA1k-based vector (Addgene ID: JPUB_000102) expressing ppdA-C and gspO genes) and three GsPilA plasmids with different or no peptide tags: - E1c - WT: pBbE1c-based vector (Addgene ID: JPUB_000098) expressing hofB-C and hofM-Q genes along with wild type GspilA) E1c - TKKTLRT: pBbE1c-based vector (Addgene ID: JPUB_000098) expressing hofB-C and hofM-Q genes along with GspilA+TKKTLRT) E1c - LRELHLNNN (pBbE1c-based vector (Addgene ID: JPUB_000098) expressing hofB-C and hofM-Q genes along with GspilA +LRELHLNNN) were tested for their ability to bind to rat tail Gibco™ Collagen I coated microplates (the protocol was adapted from Grenier (1996) [15]). The peptide tags are denoted C1 for GsPilA -TKKTLRT and C2 for GsPilA-LRELHLNNN. The results are shown below:

Figure 3. Fluorescence readings of transformed E. coli DH5alpha strains. Wells were washed three times with PBS (+0.01% TWEEN). The first four strains were added to the wells at an OD600 of 2.0. The strain expressing C2 was added at an OD600 of 0.5. Num ber of technical replicates: 4.

Table 1. Control plate fluorescence reading without washing step . Wells were not washed and contain the original culture. The first four strains were added to the wells at an OD600 of 2.0. The strain expressing C2 was added at an OD600 of 0.5. Number of technical replicates: 4.

The results from the collagen binding assay tell us that the bacteria generally bind better to the control plate than to the collagen-coated plate. The control, ΔsfmA, worked as expected, showing a low RFP signal. E.coli that was transformed with the plasmid containing the minor pilin and prepilin-peptidase genes, A1k, showed larger fluorescence transmission values compared to the bacteria that supposedly expressed wild-type pili and pili modified with the collagen 1 tag. This is probably due to the bacteria being transformed with 3 separate plasmids, expressing less RFP due to strained metabolism. E.coli transformed with the E1C-LRELHLNNN plasmid seem to be very close to the control strain ΔsfmA. Considering the strain expressing E1c-LRELHLNNN did not grow well on the plate and there is no statistical difference between the WT control and E1c-TKKTLRT follow-up experiments are needed to further characterise these parts and determine GsPilA expression.

References

[1] Zhao, W., Chen, B., Li, X., Lin, H., Sun, W., Zhao, Y., Wang , B., Zhao, Y., Han, Q. and Dai, J. (2007). Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors. Journal of Biomedical Materials Research, [online] 82A(3), pp.630–636. doi:https://doi.org/10.1002/jbm.a.31179.

[2]Wahyudi, H., Reynolds, A.A., Li, Y., Owen, S.C. and Yu, S.M. (2016). Targeting collagen for diagnostic imaging and therapeutic delivery. Journal of Controlled Release, [online] 240, pp.323–331. doi:https://doi.org/10.1016/j.jconrel.2016.01.007.

[3] Holmes DE, Dang Y, Walker DJF, Lovley DR. The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microbial Genomics. 2016 Aug 25;2(8).

[4] Vargas M, Malvankar NS, Tremblay PL, Leang C, Smith JA, Patel P, et al. Aromatic Amino Acids Required for Pili Conductivity and Long-Range Extracellular Electron Transport in Geobacter sulfurreducens. Giovannoni SJ, editor. mBio. 2013 May;4(2).

[5] Adhikari RY, Malvankar NS, Tuominen MT, Lovley DR. Conductivity of individual Geobacter pili. RSC Advances. 2016 Jan 19;6(10):8354–7.

[6] Ing NL, Nusca TD, Hochbaum AI. Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution. Physical Chemistry Chemical Physics 2017 Jan 1;19(32):21791–9.

[7] Ueki T, Walker DJF, Woodard TL, Nevin KP, Nonnenmann SS, Lovley DR. An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires. ACS Synthetic Biology. 2020 Mar 3;9(3):647–54.

[8] Nevin KP, Zhang P, Franks AE, Woodard TL, Lovley DR. Anaerobes unleashed: Aerobic fuel cells of Geobacter sulfurreducens. J Power Sources. 2011;196(18):7514–8.

[9] Dang Y, Walker DJF, Vautour KE, Dixon S, Holmes DE. Arsenic Detoxification by Geobacter Species. Liu SJ, editor. Applied and Environmental Microbiology. 2017 Feb 15;83(4).

[10] Cologgi DL, Speers AM, Bullard BA, Kelly SD, Reguera G. Enhanced Uranium Immobilization and Reduction by Geobacter sulfurreducens Biofilms. Parales RE, editor. Applied and Environmental Microbiology. 2014 Nov;80(21):6638–46

[11] Ueki T, Walker DJF, Tremblay PL, Nevin KP, Ward JE, Woodard TL, et al. Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides. ACS Synthetic Biology. 2019 Jul 12;8(8):1809–17.

[12] Eric Szmuc, David J.F. Walker, Dmitry Kireev, Deji Akinwande, Derek R. Lovley, Benjamin Keitz, Andrew Ellington. Engineering Geobacter pili to produce metal:organic filaments, Biosensors and Bioelectronics, Volume 222, 2023.

[13] Liu X, Walker DJF, Nonnenmann SS, Sun D, Lovley DR. Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens. Papoutsakis ET, editor. mBio. 2021 Aug 31;12(4).

[14] Malvankar, N.S., Vargas, M., Nevin, K., Tremblay, P.-L., Evans-Lutterodt, K., Nykypanchuk, D., Martz, E., T. Tuominen, M. and R. Lovley, D. (2015). Structural Basis for Metallic-Like Conductivity in Microbial Nanowires. American Society for Microbiology mBio, 6(2). doi:https://doi.org/10.1128/mbio.00084-15.

[15] Grenier, D. (1996). Collagen-binding activity of Prevotella intermedia measured by a microtitre plate adherence assay. Microbiology, 142(6), pp.1537–1541. doi:https://doi.org/10.1099/13500872-142-6-1537.