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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 <a href="http://parts.igem.org/Part:BBa_K2638999">BBa_K2638999</a> . | 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 <a href="http://parts.igem.org/Part:BBa_K2638999">BBa_K2638999</a> . | ||
For expression of HUHF we cloned an araBAD promoter <a href="http://parts.igem.org/Part:BBa_I0500">(BBa_I0500)</a> and a ribosome-binding site (RBS) <a href="http://parts.igem.org/Part:BBa_R0030">(BBa_R0030)</a> upstream of the CDS via BioBrick assembly. | For expression of HUHF we cloned an araBAD promoter <a href="http://parts.igem.org/Part:BBa_I0500">(BBa_I0500)</a> and a ribosome-binding site (RBS) <a href="http://parts.igem.org/Part:BBa_R0030">(BBa_R0030)</a> upstream of the CDS via BioBrick assembly. | ||
− | 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 (SDS-PAGE). | + | 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 (13000g, 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. | 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 (13000g, 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 SDS-PAGE (Fig. 1). Clearly visible bands have been recognizable at about 23 kDa, the expected hight for HUF. |
Revision as of 16:45, 7 December 2018
Improve a Part
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.
Improved Human Ferritin: BBa_K2638999
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 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).
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 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 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
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.
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