Part:BBa_K4275031
PfuFerritin-wt
Ferritins are present in a wide variety of organisms: bacteria, archaea, fungi, plants, insects and vertebrates. Their main role is presumably iron storage; excess of Fe2+ from a cell environment is internalized and then taken up by ferritin, oxidized, and stored as ferrihydride in its cavity until the cell needs it.
Ferritin has properties of an longer half life and good thermostability meaning that it can be used for nanobody displays[1]. Most importantly in our project, ferritin can be used to magnetophoresis which is plays the role of recollecting microbials in a reactive solution[3][4]. Because ferritin mainly exists in the cytoplasm of microbials, it the iron component in the cell functions to locate the bacteria in our project.
Usage and Biology
The main role of ferritin is iron storage. excess of Fe2+ from a cell environment is internalized and then taken up by ferritin, oxidized, and stored as ferrihydride[2] in its cavity until the cell needs it. Upon addition of reducing equivalents, iron is released. This function of ferritins is a key part of the homeostasis machinery of higher organisms that ensures the right balance of iron in the organism. Regulating iron levels is important given that iron needed but excess levels of iron leads to oxidative stress in higher organisms.
Characterization
Magnetic recycling
A novel design feature of our engineered E.coli was the inclusion of intracellular ferritin expression. The Fe2+ ions in ferritin allows the E. coli cells displaying cellulosome complex to be attracted by strong magnets, therefore enabling the magnetic recycling of cellulosomes to be reused.
We’ve constructed three ferritin plasmids: the ferritin wild type (Pfuferritin), the existing part (BBa_K1189065), and the wild type ferritin fused with Nb3 (Pfuferritin-Nb3) (Fig.1A). All three vectors were transformed and cultured for IPTG-inducible expression. The target proteins of all three ferritins were detected in the whole cell and supernatant samples of SDS-page (Fig.1B).
To further prove that the host E.coli cells carrying ferritin is magnetically attractive, we transformed prha-mRFP1 plasmids into competent cells containing Pfuferritin and BBa_K1189065 respectively. The co-expression of ferritin and RFP allowed magnetic recycling results to be better visualized. In both ferritin wild type and ferritin part, aggregation of red fluorescence were found near the strong magnets, proving the feasibility of magnetic recycling system (Fig.1C).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. "Fan, Kelong et al. “Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension.” Analytical chemistry vol. 90,9 (2018): 5671-5677. doi:10.1021/acs.analchem.7b05217
2. Li, Thomas L et al. “Engineering a Genetically Encoded Magnetic Protein Crystal.” Nano letters vol. 19,10 (2019): 6955-6963. doi:10.1021/acs.nanolett.9b02266
3. Tatur, Jana et al. “A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus.” Extremophiles : life under extreme conditions vol. 10,2 (2006): 139-48. doi:10.1007/s00792-005-0484-x
4. Aubry, Mary et al. “Engineering E. coli for Magnetic Control and the Spatial Localization of Functions.” ACS synthetic biology vol. 9,11 (2020): 3030-3041. doi:10.1021/acssynbio.0c00286"
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