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Team:Chiba/Project
From 2013.igem.org
Introduction
Why bother magnetizing?
We are surrounded by magnets. Magnetized materials are everywhere makes up core components of electrical switches, speaker systems, and portable memory devices (including credit cards), and diagnostic/ separation systems. Our hope is to program E. coli. cell to turn into magnets to be used for various purposes.
Magnet force is unique in that;
1. Non-contact (least chance of damaging and contamination)
2. Barrier-free mode of collections (can penetrate the physical blockage)
3. Rapidity (immediate exercise)
4. Duration (eternal action if needed)
Overview/Strategies
We have three steps to create magnetic E. coli.
1. Reprogramming the iron homeostasis:To maximize the chance of magnetization, we would like to pump as much Fe into the cell as possible, and keep it. To this end, we tried to eliminate the negative controller (encoded by fur) on the Fec system (iron importer). Also, we tried to knock down the Fe exporter fieF.
2. Establishment of Iron storage system: Fe (II) has severe toxicity to the cell because it casts multiple damages to the DNAs via Fenton reaction. To deal with this problem, we tried below thing; ferritins are cage-shaped multi-subunit proteins for collecting and accumulating ferrous ions (Fe (II)) inside. This way you can well isolate Fe from the cytosolic components.
3. Reprogramming the redox state of the cell: Some metal oxides of the spinel type i.e. Fe3O4 have ferrimagnetism due to the disparity of magnetic moment. To be better attracted by the magnet, higher ratio of Fe (III) over Fe (II) is preferred. To this end, E. coli 's cytosol system will be reprogrammed from reducing to oxidating, by knocking down genes encoding trxB and gor so that ferrous ions are easier to be oxidized and exist in cytosol.
Because most of the genetic operation above mentioned should have significant impact to the cell, we used CRISPRi technology for the temporal/ transitional knock-out of the target genes above.
Also, to speed up the construction of BioBrick, we re-engineered the existing Biobrick (BBa_I746908) coding Arabinose-triggering GFP generators so that one can quickly replace the GFP with genes of interest using GoldenGate method.
Project
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Reprogramming the iron homeostasis |
Establishment of Iron storage system |
Reprogramming the redox state of the cell |
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