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		+	==GreatBay_SCIE 2022's Characterisation==
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		+	[[Image:GreatBay SCIE--Part Fig1.png|950px|center|'''Figure 1:''' ]]
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		+	<p><h3>Fig.1</h3> The schematic representation of Fabrevivo design. Two cellulosome complexes were constructed, with cellulase and PETase subunits bind to primary scaffold CipA1B2C via type I cohesin-dockerin interaction; Attachment of CipA scaffold to OlpB-Ag3 was made possible by type II cohesin-dockerin interaction, Neae-Nb3-Ag3 association displays the whole complex on E. coli surface. Ferritin expression within E. coli host enables magnetic recycling for cellulosome complexes to be reused.</p>
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		+	[[Image:GreatBay SCIE--Part Fig2.png|950px|center|'''Figure 2:''' ]]
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		+	<p><h3>Fig.2</h3> Ferritin expression and magnetic recycling. (A) Genetic circuit construction for three types of ferritin: ferritin wild type (PfuFerritin), IGEM existing ferritin part (BBa_K1189025), and ferritin-Nb3 (PfuFerritin-Nb3) adapted for surface display system. (B) SDS-page analysis of PfuFerritin-Nb2, BBa_K1189025, Ferritin wild type (WT) respectively. (C) Magnetic recycling was conducted with Ferritin control group, RFP control group, BBa_K1189025-RFP, and Ferritin WT-RFP. Aggregation of red fluorescence can be observed in cells co-expressing ferritin and RFP. </p>
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	==Contribution (Waterloo iGEM 2020)==		==Contribution (Waterloo iGEM 2020)==

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Revision as of 12:26, 12 October 2022

Heavy chain human ferritin

This part is the heavy ferritin subunit from human ferritin, inspired by P02794 [UniParc]. Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron. This part, along with the light ferritin subunit, form a 24 multimeric iron sequestering nanoparticle (Chasteen et al., 1991). The difference between light ferritin is that this chain contains a ferroxidase centre. Protein domains which orient toward the core of ferritin molecules cause the oxidation of intracellular iron from Fe^2+ to Fe^3+ to initiate formation of a ferrihydrite core. (Chasteen et al., 1999). These nanoparticles are robust, remain stable at extreme pHs, and withstand temperature variations (Kim et al., 1999).

Ferritin's utility in iGEM

Ferritin as a nanoparticle is interesting for other iGEM teams for two reasons. Firstly, its iron core can be replaced with other compounds to serve different functions. The iGEM Calgary 2013 demonstrated this by chemically modifying recombinant ferritin's iron core into a robust colourmetric reporter. Other intriguing applications include making ferritin’s iron core magnetically active as magnetoferritin (Jordan et al. 2013), using ferritin as a nanocage for other metals, or the incorporation of other reporters such as quantum dots (Naito et al. 2013) (Figure 2).

Secondly, the ferritin nanoparticle is useful for iGEM teams as a self-assembling and spherical protein scaffold. Each of the 24 subunits forming ferritin can be fused to proteins of interest, such that when the nanoparticle assembles, proteins surround the ferritin sphere (Kim et al., 2011). The iGEM Calgary 2013 team demonstrated this by binding DNA sensing proteins, TALEs, as part of their FerriTALE sensor. The Calgary team also constructed ferritin subunits with a coiled-coil linker system so that other teams can scaffold proteins to E-coil ferritin (BBa_K1189018, BBa_K1189019, BBa_K1189020, BBa_K1189037). See Figure 3 for a demonstration of these applications.

References

Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.

Clavijo Jordan, V., Caplan, M. R., & Bennett, K. M. (2010). Simplified synthesis and relaxometry of magnetoferritin for magnetic resonance imaging. Magnetic Resonance in Medicine, 64(5), 1260-1266.

Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.

Naito, M., Iwahori, K., Miura, A., Yamane, M., & Yamashita, I. (2010). Circularly polarized luminescent CdS quantum dots prepared in a protein nanocage. Angewandte Chemie International Edition, 49(39), 7006-7009.

GreatBay_SCIE 2022's Characterisation

Fig.1

The schematic representation of Fabrevivo design. Two cellulosome complexes were constructed, with cellulase and PETase subunits bind to primary scaffold CipA1B2C via type I cohesin-dockerin interaction; Attachment of CipA scaffold to OlpB-Ag3 was made possible by type II cohesin-dockerin interaction, Neae-Nb3-Ag3 association displays the whole complex on E. coli surface. Ferritin expression within E. coli host enables magnetic recycling for cellulosome complexes to be reused.

Fig.2

Ferritin expression and magnetic recycling. (A) Genetic circuit construction for three types of ferritin: ferritin wild type (PfuFerritin), IGEM existing ferritin part (BBa_K1189025), and ferritin-Nb3 (PfuFerritin-Nb3) adapted for surface display system. (B) SDS-page analysis of PfuFerritin-Nb2, BBa_K1189025, Ferritin wild type (WT) respectively. (C) Magnetic recycling was conducted with Ferritin control group, RFP control group, BBa_K1189025-RFP, and Ferritin WT-RFP. Aggregation of red fluorescence can be observed in cells co-expressing ferritin and RFP.

Contribution (Waterloo iGEM 2020)

Summary: While the heavy and light chain human ferritin subunits have been added to the iGEM parts registry with adequate description of their function, we wanted to compare them more thoroughly. Therefore, our discussion includes both chains’ stability and ability to uptake iron.

Documentation: Human ferritin heteropolymers can be composed with varying ratios of heavy (H) and light (L) chains, and different forms exist naturally depending on the tissue examined (Boyd et al., 1985). However, the knowledge of the two chains is not sufficient enough to predict the properties of any given heteropolymer.

Homopolymers composed of strictly light or heavy chains can be synthesized, which can be advantageous as heavy and light chains have different properties: 1. H-rich ferritins turn over more rapidly than L-rich ferritins 2. H-rich ferritins accumulate in iron-rich tissues 3. L-rich ferritins take up and release iron more rapidly than L-rich ferritins

Within the shell, H and L subunits are interchangeable as the key residues are conserved.

L subunits contain 15 hydrophilic iron binding residues, while only 7 of these exist in H subunits. This results in a reduced ability to nucleate iron in H subunits, causing faster iron uptake and release as well as less iron accumulation in H-rich ferritins.

Ferritin’s uptake of iron is either driven by H-chain ferroxidase activity (Levi et al., 1988) or driven by iron autoxidation which occurs in the absence of H-chains.

Stability:

• In vitro, H ferritin oxidizes iron at a much faster rate than L ferritin because of the presence of a particular ferroxidase centre in H ferritin that is absent in L ferritin (Santambrogio et al., 1993)
• L ferritin induces iron mineralization more efficiently than H ferritin (Santambrogio et al., 1993)
• L ferritin is able to withstand physical denaturation better than H ferritin because of the replacement of the ferroxidase centre of the H chain with a salt bridge in the L-chain (Santambrogio et al., 1993)

References:

Boyd, D., Vecoli, C., Belcher, D.M., Jain, S.W., & Drysdale, J.W. (1985). Structural and Functional Relationships of Human Ferritin H and L Chains Deduced from cDNA Clones. The Journal of Biological Chemistry, 260(21). 11755-11761.

Levi, S. et al. (1988). Mechanism of Ferritin Iron Uptake: Activity of the H-chain and Deletion Mapping of the Ferro-oxidase Site. The Journal of Biological Activity, 263(34). 18086-18092.

Santambrogio, P. et al. (1993). Production and Characterization of Recombinant Heteropolymers of Human Ferritin H and L Chains. The Journal of Biological Chemistry, 268(17). 12744-12748.

Sequence and Features