Team:ManhattanCol Bronx/Design

Project Design

We designed our project in terms of three specific aims, each of which were meant to additively increase the efficiency of a bioanode. These three aims include:

◙ Improving the activity and efficiency of the electron producing enzyme, glucose oxidase (GOx). We used mutant variants of the Aspergillus niger GOx that were reported to have increased stability and activity (1). We then introduced a cysteine tag to the GOx variants as a way to sequester them to our anode.

◙ Increasing the anode surface area as a means of increased affinity for GOx and a decreased distance that an electron must travel to reach the anode (direct transfer of electrons to the anode)

◙ Creating an electric E. coli strain that harbors the cytochrome electron transport system of Shewanella oneidensis. S. oneidensis utilizes a pathway of electron transfer enzymes that ultimately reduce extracellular metal ions during anaerobic respiration. We intend to express the S. oneidensis cytochrome operon in E. coli to create an E. coli strain that can directly transfers electrons to an anode instead of metal ions.

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gBlock Design & Cloning

Glucose Oxidase (GOx) Variants:

We derived the sequence for A. niger GOx from the NCBI Genebank number AAA32695.1(E.C 1.1.3.4). This sequence was utilized by Holland et. al. (2) for the rational design of increased stability in GOx mutants. Each of our GOx genes was derived from this wild type (Wt) sequence. Taking advantage of IDT’s generous support of iGEM we ordered gBlock fragments for each of our constructs. These were designed for cloning into both our expression plasmid (Novagen, pCDF-duet) and the biobrick backbone pSB1C3.

GOx Wt brick design

Initially, we designed the Wt GOx amino acid sequence based on that found through NCBI to create a DNA sequence that was codon optimized for E. coli (codon optimization done through IDT). To this sequence we then added an N-terminal 6x histidine tag followed by a TEV protease site. These coding regions were added to install an affinity tag for the purpose of protein isolation on a nickel-resin column that could then be released following purification. Next, we considered a short C-terminal glycine linker followed by a SacI site just prior to the stop codon. We rationalized that this linker would allow for enzymatic addition of future protein fusions or tags that proved beneficial for specific assays. Each of the terminal sequences were also codon optimized for E. coli. Finally, we added the required biobrick prefix and suffix sequences.

Once the DNA sequence was finalized and matched our expected reading frame we then screened it for any illegal biobrick sequences. When the following sites were found: EcoRI, XbaI, SpeI, and PstI; they were removed by using silent mutations that destroyed the enzymatic restriction recognition site but retained the coding for the amino acid.

Each GOx mutant was then designed starting from the Wt gBlock unit.

GOx-4mut brick design

The Quadruple GOx mutant (GOx-4mut) was derived from research by Holland et al. (3) that was found to have increased stability, activity, and possess the ability to directly transfer electrons to a gold nanoparticle. The points of mutation in the original research were T56V, T132S, H447C, and C521V. Each of the mutants were designed into our gBlock by using the codon optimized sequence for Val, Ser, and Cys. The final gBlock sequence for the quadruple mutant matched that of the Wt gBlock, except for the point mutations. The sequence was checked for illegal sites as stated above.

GOx-cys and 4mut-cys design

Our final two GOx enzymes were derivatives of the GOx-Wt and the GOx-4mut gBlocks. One enzyme was simply GOx-Wt with a C-terminal 3x cysteine tag (GOx-cys) . This sequence was added as an affinity tag for our anode due to reports suggesting that gold nanoparticles are attracted to thiol groups (4,5). The last GOx derivative was GOx-4mut with the addition of a C-terminal 3x cysteine tag (GOx-4mut-cys). These two tagged mutants were designed in an effort to create a sequestering mechanism our gold anode. As stated above, each construct was codon optimized for E. coli codons and scanned for illegal sites prior to IDT ordering.

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Glucose Oxidase (GOx) Variant protein expressions:

Our cloning efforts utilized the biobrick prefix and suffix installed EcoRI and PstI sites, respectively, for ligation insertion into both the pCDF-duet and pSB1C3. The pCDF plasmid utilizes a T7lac promoter that is IPTG inducible. With these constructs we expressed our variants and then passed the lysates of these expressions over a Ni²⁺ column for protein purification. 5, 1 ml elutions were collected and each was tested for GOx activity using an AMPLEX Red GOx kit (Invitrogen). GOx variants that showed activity were then used for the oxidation reaction on our gold anode.

mtrCAB and CymA:

The mtrCAB operon and CymA gene are responsible for electron transport across the cell membrane in the electric bacteria Shewanella oneidensis (6,7). The mtr operon and CymA gene express a system of cytochromes that work together to pass electrons to the extracellular matrix to reduce metals (or an anode). CymA is an inner membrane tetraheme cytochrome c (7). MtrA is a periplasmic decaheme cytochrome c and MtrC is an extrcellular decaheme cytochrome c (8). Based on previous literature (9) expressing CymA in E. coli, in addition to the mtrCAB operon, increases the reduction rate of extracellular acceptors.

Our E.(lectro) coli project was conceived with the idea of stably expressing the mtrCAB operon and the CymA gene simultaneously in E. coli cells. While previous teams (2014 TU_Delft-Leiden) have worked toward expressing the mtrCAB operon we set out to improve their previous brick, K1316012 by coupling it to CymA and also exchanging the promotor. This brick is expected to increase the efficiency of the Shewanella electron transport in E. coli. Unfortunately, we were unsuccessful in our attempts to clone this brick and are vehemently working toward this goal. This cloning will not be completed by the deadline of this years competition. However, we will continue to work on building this construct because we feel this portion of our project is potentially an important step towards creating a more efficient bioanode.

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nANODE Design and Set Up

Design

To design the nANODE, we applied a template based synthesis of Gold nanowires (Au-NW). The rationale behind using nanowires as opposed to a flat Au surface is two fold. First, the nanowires have an increased surface area in comparison to the flat anode. This is expected to increase the area for GOx adsorption onto the surface of the anode. Our hypothesis is that there is a direct correlation between the amount of enzyme in close proximity (on the surface) to the anode and the performance of the anode in a Biofuel Cell. The second reason for using nanowires as opposed to a flat anode or nanoparticles, is the directionality of the nanowires. To understand why, we should look to how electricity is transported in the real world. To bring the power into our homes from power plants, the electricity is transmitted through wires . Additionally, to transport electricity from outlets to other devices, we again turn to the use of wires to move charge from one place to another. It only makes sense that we should utilize the same concepts within our device toe more efficiently and effectively deliver electricity from the enzymes to the anode and the rest of the cell. This leads us to our second hypothesis; the 1 dimensional (1D) nature of the nanowires will greatly enhance the removal of charge from the anode, which is suspected to enhance the efficiency of the overall device.

Set Up

To achieve our goals set forth in our design, we utilized a template based "U-Tube" (not YouTube!!) synthetic method to create Au-NW.

This method was selected for several reasons. First, by using a template we can physically control the size of the Au-NW, allowing us to tailor the design of the anode for optimal performance. Secondly, this reaction occurs at room temperature, in a relatively benign solvent of Ethanol. Thirdly, we can easily remove the template and fully characterize the properties of the Au-NWs. Finally, by carefully controlling the reaction conditions, a free standing array of Au-NWs can be produced. This final reason is very exciting for our application because it has the potential to make "nanograss". (10)

It is our hypothesis that this nanograss will be very advantageous for use in the final device. When the Au-NWs are removed from the template and placed onto the anode, they look like "pick-up sticks" (google it!). This orientation, while better than a flat anode, is expected to be less ideal than the "nanograss" orientation. We reason that the vertical alignment within the "nanograss" sample will greatly enhance the removal of charge from the enzyme to the anode, whilst maintaining a large surface area.

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References

1. Prabhulkar, S., Tian, H., Wang, X., Zhu, J-J. and Li, C-Z. Engineered proteins: Redox properties and their applications. Antioxidants and Redodox Signalling, 2012, vol. 17 (12), pp. 1796-1822.
2. Holland, T.J., Harper, J.C., Dolan, P.L., Manginell, M.M., Arango, D.C., Rawlings, J.A., Apblett, C.A., and Brozik, S. Rational redesign of glucose oxidase for improved catalytic function and stability. PLoS ONE, 2012, vol. 7 (6), pp. e37924-10.
3. Holland, J.T., Lau, C., Brozik, S., Atanassov, P., and Banta, S. Engineering of glucose oxidase for direct electron transfer via site-specific gold nanoparticle conjugation. 2011, J. Am. Chem. Soc. 133 (48), pp. 19262-19265.
4. Nuzzo, R.G., and Allara, D.L. Adsorption of bifunctional organic disulfides on gold surfaces. 1983, J. Am. Chem. Soc. 105, pp. 4481–4483.
5. Karyakin, A. A., Presnova, G. V., Rubtsova, M. Y., and Egorov, A. M. Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups. 2000, Anal. Chem. 72, pp. 3805–3811.
6. Shi, L., Rosso, K. M., Clarke, T. A., Richardson, D. J., Zachara, J. M., and Fredrickson, J. K. Molecular underpinnings of Fe(III) oxide reduction by Shewanella oneidensis MR-1. 2012, Front. Microbiol. 3, pp. 50.
7. Marritt, S. J., McMillan, D. G. G., Shi, L., Fredrickson, J. K., Zachara, J. M., Richardson, D. J., Jeuken, L. J. C., and Butt, J. N. The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1. Biochem. 2012, Soc. Trans. 40, pp. 1217−1221.
8. Fredrickson, J. K., Zachara, J. M., Kennedy, D. W., Dong, H., Onstott, T. C., Hinman, N. W., and Li, S.-M. Biogenic iron mineralization accompanying the dissimilatory reduction of hydrous ferric oxide by a groundwater bacterium. 1998, Geochim. Cosmochim. Acta. 62, pp. 3239−3257.
9. Jensen, H.M.,TerAvest, M.A., Kokish, M.G., and Ajo-Franklin, C.M. CymA and exogenous flavins improve extracellular electron transfer and couple it to cell growth in Mtr-expressing escherichia coli. 2016, ACS Synth. Biol. 5 (7), pp. 697-688
10. Koenigsmann, C., Santulli, A.C., Sutter, E., Wong, S.S. Ambient Surfactantless Synthesis, Growth Mechanism, and Size-Dependent Electrocatalytic Behavior of High-Quality, Single Crystalline Palladium Nanowires. 2011, ACS Nano. 5 (9), pp 7471–7487