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Short summary

The Human Ferritin Heavy Chain (HUHF) BBa_K2638999 was successfully cloned and expressed in Escherichia coli DH5 alpha. After protein purification HUHF was used to produce gold and silver nanoparticles which was ensured by examinations with the Transmission Electron Microscope and Energy-dispersive X-ray spectroscopy (EDX). Thus, we improved BBa_K1189019 which is not able to form gold and silver nanoparticles.

The Calgary 2013 iGEM team used the human ferritin wildtype (BBa_K1189019) as reporter protein for a test strip. They expressed the human ferritin heavy and light chain heterologous using Escherichia coli. In the cells, the ferritin produced its characteristic iron core, which was colored with the help of fenton chemistry to produce the prussian blue iron complex. Beside the function as reporter, the team mentioned the capability of ferritin to produce nanoparticles from other metal ions.

**Figure 1:** Ferritin is suitable for metal recycling, since it can form e.g. iron, silver and gold nanoparticles in its cavity.

The capability of human ferritin to bind different metal ions and form nanoparticles makes it suitable for the recycling of different valuable metal ions (Ensign et al., 2004; Domínguez-Vera et al., 2007). In addition, nanoparticles formed inside/by ferritin have advantages over nanoparticles produced by conventional methods. On the one hand the ferritin encapsulated nanoparticles are water soluble due to the protein shell. On the other hand the maximal inner diameter of ferritin of 8 nm causes a upper size restriction of the nanoparticles inside the ferritin (Butts et al., 2008). This restriction is desirable for various applications (Castro et al., 2014).

The wildtype ferritin has reactive amino acids on the outside and inside of the protein shell, causing nanoparticle synthesis at both surfaces. An optimization of the wildtype human ferritin towards a nanoparticle syntheses mainly in the interior can therefore favor a unified production of different nanoparticles (Butts et al., 2008).

Improved Human Ferritin: BBa_K2638999

To improve the ferritins capability to direct metal ions to its inside and to increase its ability to form gold and silver nanoparticles, we constructed a mutated version of the human ferritin heavy chain (HUHF) (BBa_K2638999). Following Christopher A. Butts et al. (2008) we removed reactive cysteine and histidine residues from exterior of the HUHF and added additional cysteine residues at the interior. This way the production of nanoparticles at the exterior surface is prevented or at least decreased.

**Figure 2:** Alignment of the protein sequences of the wildtype and the mutated human ferritin heavy chain. The Alignment was produced with Clustal Omega (Goujon et al., 2010, Sievers et al., 2011).

In Figure 2 the amino acid sequence alignment of the wildtype human ferritin and the mutated human ferritin is shown. The exterior residues C91R, C103A, C131S, H14D and H106Q, and the interior residues E65C, E141C, E148C, K87Q and K144C were mutated. The mutations have no influence on the structure of the ferritin as shown in Figure 3.

**Figure 3:** Protein structures of the wildtype human ferritin (A, RCSB ID 4oYN) and the mutated human ferritin (B, RCSB ID 3ES3). Despite the mutations of ten amino acids the ferritin retains its shape. The protein structeres were generated with Chimera (Pettersen et al., 2004).

We ordered the CDS for the Human Ferritin Heavy Chain (HUHF) as gBlock from IDT. Due to the complications of the synthesis of HUHF l, we had to split the protein into three parts with 50 basepairs overlapping sequences to each other and to the backbone pSB1C3. This enables combined cloning of all three HUHF-gBlocks by Gibson Assembly into the plasmid backbone of pSB1C3, thus creating the basic part BBa_K2638999 . For expression of HUHF we cloned an araBAD promoter (BBa_I0500) and a ribosome-binding site (RBS) (BBa_R0030) upstream of the CDS via BioBrick assembly. HUHF was expressed in E. coli DH5α. Therefore, 50 mL LB cultures in shaking flasks were inoculated from an overnight culture to achieve an OD₆₀₀ of 0.1. The cultures were cultivated at 37 °C and 140 rounds per minute (rpm). After growing to an OD₆₀₀ of 0.6-0.8, HUHF expression was induced with 1 % L-arabinose. After induction the flasks were incubated at 28 °C and 140 rpm for six hours. Samples were taken hourly for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the samples were further purified. Therefore, the protocol of Dr. Jon Marles Wright has been applied. The samples were pelleted (13000 g, 1 min), the media was discarded and cell pellets were resuspended in buffer A (50 mM tris pH 8, 1 mM DTT, 0.1 mM ethylenediaminetetraacetic acid, 20 mM mannitol). Afterwards the cells were lysed by sonication, centrifuged (10000 g, 20min), heated to 80 °C for 10 minutes, put on ice for 10 minutes and centrifuged (10000 g, 10 min) again. Purified HUHF was located in the supernatant. HUHF purified by this workflow was analyzed with a SDS-PAGE (Fig. 1). Clearly visible bands have been recognizable at about 23 kDa, the expected hight for HUF.

**Figure 4:** SDS-PAGE of purified Human Ferritin Heavy Chain (BBa_K2683999). HUHF under control of the araBAD promoter (BBa_I0500) was expressed in *E. coli* DH5 alpha and afterwards purified. Shown are samples were expression was induced with 1 % arabinose (third, sixth ninth and twelfth lane) and controls without induction (second, fifth, eighth and eleventh lane) as well as empty-vector controls (pSB1C3 without insert, first, fourth, seventh and tenth lane).

The SDS-PAGE

of the purified HUHF showed intense bands at about 25 kDa, fitting to the theoretical molecular weight of HUHF of 24.087 kDa. To ensure the identity of the HUHF the band was cut out of the SDS-PAGE and prepared for matrix assisted laser desorption ionisation with time of flight analysis (MALDI-TOF) measurements. These show distinctive peaks for the HUHF. In addition, tandem mass spectrometry measurements show peptides specific for HUHF, thereby confirming HUHF expression as well as a successful purification workflow. Besides, measurements reveal that the HUHF has been expressed.

**Figure 5:** Results of MALDI-TOF measurements. The peptide fragments show a recognizable pattern for the HUHF.

In Figure 5 the specific peptide fragments pattern of the HUHF can be seen. The samples were digested with trypsin prior MALDI-TOF. Per tandem mass spectrometry the peak with the m/z = 1345.649 was further examined (Figure 6).Thus, all results confirm that the measured sample contains HUHF.

**Figure 6:** Results of the tandem mass spectrometry of the peak 1345 of HUHF.

The purified HUHF was used to produce gold and silver nanoparticles. Therefore, HUHF samples were prepared by removing the ferritin bound Fe³⁺ ions. By applying the (name of the protocol) protocol, the Fe³⁺ ions have been reduced to Fe²⁺ ions. After the iron ions were removed, AuHCl₄ and AgNO₃ solutions were added as stated in the (name of the protocol) protocols. The HUHF with the AuHCl₄ solution was incubated for 1.5 hours whereas the AgNO₃ solution was incubated for 18 hours while being illuminated by a 60 watts lamp. Afterwards the samples were centrifuged (10.000 g, 10 min) and further purified with a 100 kDa protein columns to remove denaturated HUHF. For demonstration of nanoparticle formation and determination of nanoparticle composition the samples were examined using a transmission electron microscope (TEM).

Analysis workflow: The TEM images of the nanoparticles were automatically analyzed in ImageJ. The particles were separated from the background by k-means clustering. Afterwards touching particles were segmented by watershed segmentation. The processed image was binarized and a particle analysis was performed.

**Figure 7:** Automatic identification of 147 silver nanoparticles in the wildtype human ferritin sample (BBa_K1189019).

Figure 7 shows a TEM image with 147 identified silver nanoparticles produced by the wild type human ferritin (BBa_K1189019). The particles are between 24.5 and 1597.8 nm in size with one very big particle with a size of 7272.3 nm, which seems to consist in many agglutinated silver nanoparticles. No particle was found in the expected size of about 8 nm.

**Figure 8:** Automatic identification of 708 silver nanoparticles in the gold silver mutant ferritin sample (BBa_K2638999). 431 (60.8%) of the nanoparticles had a mean diameter of 8 nm or less.

Figure 8 shows a TEM image with 708 identified silver nanoparticles produced by the gold silver mutant ferritin sample (BBa_K2638999). The particles have a size between 1.8 and 34.8 nm. 120 of the silver nanoparticles (16.9 %) are exactly in the expected size of 7 to 9 nm which indicates that at least all of these particles are produced by our improved ferritin (BBa_K2638999).

**Figure 9:** The silver nanoparticles in our gold silver mutant ferritin (BBa_K2638999) with a mean diameter of 8.2 nm were significant smaller than the nanoparticles of the wildtype human ferritin (BBa_K1189019) with a mean diameter of 531.8 nm.

The direct comparison of our new gold silver mutant ferritin (BBa_K2638999) and the old wild type human ferritin (BBa_K1189019) in figure 9 shows that our improved enzyme produces nearly five times more silver nanoparticles which are 98.5 % smaller than the silver nanoparticles produced by the wild type ferritin. This proves that the new ferritin enzyme is much more suitable for producing silver nanoparticles than the wild type version.

**Figure 10:** Automatic identification of 2 gold nanoparticles (13 and 10 nm) in the wildtype human ferritin sample (BBa_K1189019).

Figure 10 shows two gold nanoparticles of 13 and 10 nm diameter that were produced by the wild type human ferritin sample (BBa_K1189019). They are slightly bigger than the expected size between 7 and 9 nm, and thus it can’t be ensured that these particles are really produced by that enzyme.

**Figure 11:** Automatic identification of 2 gold nanoparticles (7 and 9 nm) in the gold silver mutant ferritin sample (BBa_K2638999). These nanoparticles are approximately 30 % smaller than the nanoparticles produced by the wildtype ferritin.

Figure 11 shows two gold nanoparticles of 7 and 9 nm diameter that were produced by the gold silver mutant ferritin (BBa_K2638999). They are exactly in the expected size range, although it is difficult to draw reliable conclusions from this small size and number of particles.

Outlook

We could show that commercially available metal nanoparticles can be printed and melted to produce electronic circuits using temperatures as low as 350°C compared to the very high melting temperature of 1083°C required to melt bulk copper. To use the NP we produced ourself for the printing of conductive circuits would have required higher amounts of the synthesized nanoparticles and would also have required further purification steps. Through our talks to Benjamin Lehner working at NASA we knew already that proteins mixed with the nanoparticles would lead to no or very poor conductivity when trying to sinter them without further purification. Using our own nanoparticles would be the logical next step.

**Figure 12:** Commercially available copper nanoparticles of 40 nm size were prepared under a nitrogen (N₂) gas atmosphere in a rather simple setup using a plastic bag with inlets for the hands. The NPs where enclosed by putting two microscope slides on top of each other to prevent oxygen from reaching the NP during the sintering process. The NPs between the glass slides were heated to 350 °C for 10 minutes. The heating step was repeated 3 times resulting in a solid surface with a measurable conductivity of around 0.9 Megaohm. Heating experiments at lower temperatures of only 250 °C showed no conductivity. Also heating to 350 °C under an oxygen atmosphere (no glas slide was put on top) only lead to structures which where not conductive. We also tried to solve the NP in water which was deoxidized by bubbling N₂ gas through it for 20 minutes. The NP solved in water and later dried under the N₂ atmosphere also showed no conductivity after treating them at 350 °C despite putting them between glass slides to avoid the oxidising effects of air. We suspect the NP reacted and oxidized when being treated with water which would result in a much higher melting temperature.

Nanoparticles produced with ferritin can also be used in various other applications (Figure 13). As example, they can be directly used inside the ferritin cage for molecular imaging (Wang et al., 2017b). When extracted, they can be used as antibacterial agent, in particular silver nanoparticles (Wang et al., 2017a) and as biosensors (Castro et al., 2014)

**Figure 13:** Possible applications of nanoparticles produced with ferritin.

Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
Butts, C.A., Swift, J., Kang, S., Di Costanzo, L., Christianson, D.W., Saven, J.G., and Dmochowski, I.J. (2008).. Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein † , ‡. Biochemistry 47: 12729–12739.
Castro, L., Blázquez, M.L., Muñoz, J., González, F., and Ballester, A. (2014).. Mechanism and Applications of Metal Nanoparticles Prepared by Bio-Mediated Process. Rev. Adv. Sci. Eng. 3.
Ensign, D., Young, M., and Douglas, T. (2004).. Photocatalytic synthesis of copper colloids from CuII by the ferrihydrite core of ferritin. Inorg. Chem. 43: 3441–3446.
Goujon, M., McWilliam, H., Li, W., Valentin, F., Squizzato, S., Paern, J., and Lopez, R. (2010).. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38: W695-699.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004).UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612.
Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., and Higgins, D.G. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7: 539.
Ummartyotin, S., Bunnak, N., Juntaro, J., Sain, M., and Manuspiya, H. (2012). . DSynthesis of colloidal silver nanoparticles for printed electronics. /data/revues/16310748/v15i6/S1631074812000549/.
Wang, L., Hu, C., and Shao, L. (2017a).. The antimicrobial activity of nanoparticles: present situation and prospects for the future. Int. J. Nanomedicine 12: 1227–1249.
Wang, Z., Gao, H., Zhang, Y., Liu, G., Niu, G., and Chen, X. (2017b).. Functional ferritin nanoparticles for biomedical applications. Front. Chem. Sci. Eng. 11: 633–646.

@@ Line 119: / Line 119: @@
 HUHF was expressed in <i>E. coli</i> DH5α. Therefore, 50 mL LB cultures in shaking flasks were inoculated from an overnight culture to achieve an OD<sub>600</sub> of 0.1. The cultures were cultivated at 37 °C and 140 rounds per minute (rpm). After growing to an OD<sub>600</sub> of 0.6-0.8, HUHF expression was induced with 1 % L-arabinose. After induction the flasks were incubated at 28 °C and 140 rpm for six hours. Samples were taken hourly for sodium dodecyl sulfate polyacrylamide gel electrophoresis <a href="https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--SDS_PAGE_LK.pdf">(SDS-PAGE)</a>.
 Moreover, the samples were further purified. Therefore, the <a href="https://static.igem.org/mediawiki/2018/7/7e/T--Bielefeld-CeBiTec--Ferritin_Purification_LK.pdf">protocol</a> of <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Attributions">Dr. Jon Marles Wright</a> has been applied.  The samples were pelleted (13000 g, 1 min), the media was discarded and cell pellets were resuspended in buffer A (50 mM tris pH 8, 1 mM DTT, 0.1 mM ethylenediaminetetraacetic acid, 20 mM mannitol). Afterwards the cells were lysed by sonication, centrifuged (10000 g, 20min), heated to 80 °C for 10 minutes, put on ice for 10 minutes and centrifuged (10000 g, 10 min) again. Purified HUHF was located in the supernatant.
-HUHF purified by this workflow was analyzed with a SDS-PAGE (Fig. 1). Clearly visible bands have been recognizable at about 23 kDa, the expected hight for HUF.
+HUHF purified by this workflow was analyzed with a <a href="https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--SDS_PAGE_LK.pdf">SDS-PAGE</a> (Fig. 1). Clearly visible bands have been recognizable at about 23 kDa, the expected hight for HUF.
@@ Line 135: / Line 135: @@
 <article>
-The SDS-PAGE of the purified HUHF showed intense bands at about 25 kDa, fitting to the theoretical molecular weight of HUHF of 24.087 kDa. To ensure the identity of the HUHF the band was cut out of the SDS-PAGE and prepared for matrix assisted laser desorption ionisation with time of flight analysis (MALDI-TOF) measurements. These show distinctive peaks for the HUHF. In addition, tandem mass spectrometry measurements show peptides specific for HUHF, thereby confirming HUHF expression as well as a successful purification workflow. Besides, measurements reveal that the HUHF has been expressed.
+The <a href="https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--SDS_PAGE_LK.pdf">SDS-PAGE</a> of the purified HUHF showed intense bands at about 25 kDa, fitting to the theoretical molecular weight of HUHF of 24.087 kDa. To ensure the identity of the HUHF the band was cut out of the <a href="https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--SDS_PAGE_LK.pdf">SDS-PAGE</a> and prepared for matrix assisted laser desorption ionisation with time of flight analysis (MALDI-TOF) measurements. These show distinctive peaks for the HUHF. In addition, tandem mass spectrometry measurements show peptides specific for HUHF, thereby confirming HUHF expression as well as a successful purification workflow. Besides, measurements reveal that the HUHF has been expressed.
 </article>
@@ Line 237: / Line 237: @@
   <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/3/32/T--Bielefeld-CeBiTec--cg--PrintingElectronics.png">
   <figcaption>
-  <b>Figure 12: </b>Commercially available copper nanoparticles of 40nm size were prepared under a nitrogen (N2) gas atmosphere in a rather simple setup using a plastic bag with inlets for the hands. The NPs where enclosed by putting two microscope slides on top of each other to prevent oxygen from reaching the NP during the sintering process. The NPs between the glass slides were heated to 350°C for 10 minutes. The heating step was repeated 3 times resulting in a solid surface with a measurable conductivity of around 0.9 Megaohm. Heating experiments at lower temperatures of only 250°C showed no conductivity. Also heating to 350° under an oxygen atmosphere (no glas slide was put on top) only lead to structures which where not conductive. We also tried to solve the NP in water which was deoxidized by bubbling N2 gas through it for 20 minutes. The NP solved in water and later dried under the N2 atmosphere also showed no conductivity after treating them at 350°C despite putting them between glass slides to avoid the oxidising effects of air. We suspect the NP reacted and oxidized when being treated with water which would result in a much higher melting temperature.
+  <b>Figure 12: </b>Commercially available copper nanoparticles of 40 nm size were prepared under a nitrogen (N<sub>2</sub>) gas atmosphere in a rather simple setup using a plastic bag with inlets for the hands. The NPs where enclosed by putting two microscope slides on top of each other to prevent oxygen from reaching the NP during the sintering process. The NPs between the glass slides were heated to 350 °C for 10 minutes. The heating step was repeated 3 times resulting in a solid surface with a measurable conductivity of around 0.9 Megaohm. Heating experiments at lower temperatures of only 250 °C showed no conductivity. Also heating to 350 °C under an oxygen atmosphere (no glas slide was put on top) only lead to structures which where not conductive. We also tried to solve the NP in water which was deoxidized by bubbling N<sub>2</sub> gas through it for 20 minutes. The NP solved in water and later dried under the N<sub>2</sub> atmosphere also showed no conductivity after treating them at 350 °C despite putting them between glass slides to avoid the oxidising effects of air. We suspect the NP reacted and oxidized when being treated with water which would result in a much higher melting temperature.
   </figcaption>
   </figure>