Background
Gene regulation has typically employed chemically mediated expression systems, which are slow, taking on the order of hours to switch on gene expression and up to days to switch off as drugs are eliminated [1]. Though genetically encoded light sensors have provided a robust and convenient way to spatiotemporally control gene expression [2], light delivery has limited penetration. Means for temporally regulating gene expression with minimal perturbation is in demand [3].
Low and medium electromagnetic wave can penetrate deep tissues with minimal energy absorption [4]. However, induced by electromagnetic signal, magnetic nanoparticles can absorb energy and heat [5]. Properties of electromagnetic wave and magnetic particles provide possibilities of remote regulation.
Sarah A Stanley et al. [3]have successfully constructed remote regulation system in mice (Fig.1.), based on ferritin-TRPV1 system. Ferritin is a kind of iron-storage protein in organisms, which could synthesize ferric oxihydroxide core in its hollow protein shell [6]. TRPV1 is a kind of temperature-sensitive channel: When local temperature rises, TRPV1 gates calciumto activate a Ca2+-sensitive promoter [4]. However, TRPV1 has limited use in Prokaryote. Thus, we designed Magthermo coli—a platform for remote regulation of gene expression by electromagnetic signal in E.coli.
Overview
How does it work?
There are two main components in our magthermo coli: magnetic receiver & thermosensitive regulator. Inducing with electromagnetic field，magnetic receiver would be heated, thus raise the ambient temperature. In response to the change of temperature, thermosensitive regulator would initiate the downstream gene (GFP for example) expression.
Magnetic Receiver
Magnetic nanoparticles can serve as a nanosource of heat [5]. That’s why we choose Ferritinas magnetic receiver: Ferritin is a kind of iron-storage protein in many organisms, which could synthesizeferric oxihydroxide core in its hollow protein shell [6] (Fig.2.). Once exposed to electromagnetic field, the ferric oxihydroxide core will be heated, raising the ambient temperature.
Thermosensitive Regulator
For thermosensitive regulator，we choseRNA thermometer and designed a thermosensitive T7 RNA polymerase. RNA thermometer is a structured RNA which could expose SD sequences only at appropriate temperature [7] (Fig.3.). Thermosensitive T7 RNA polymerase is a T7 RNA polymerase interrupted by a temperature-sensitive intein. Temperature-sensitive Intein is a kind of polypeptide that could self-splice and ligate it’s flanking polypeptides at specific temperature. Thus, interrupted T7 RNA polymerase can ininate the downstream signaling [8] (Fig.4.).
Measuring Technique
In mag-receiver section:
(1)We found it complex and costly to measure magnetism of E.coli, thus, constructed a device for easy measure of magnetism—Captor (Fig.5.).
(2)During verification for in vivo mineralization of ferritin, we found little literatures offering accurately detecting method. Thus, we referenced method in vitro[9] and successfully detected iron core in ferrin after in vivo mineralization.
(3)To supply ourmodeling with concentration of ferritin per cell, we explored a method for valuing protein concentration per cell, and tried to make it more convenient.
In thermo-regulator section:
(1)We explored the measurement method to identify the efficiency of thermosensitive regulator under heat stress.
(2)During testing PBAD, we found it inconvenient to take photos of different plates and comparing them. Thus, we extended the function of Captor, and made it convenient for testing optimal inducement concentration on plates (Fig.6.).
References
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2.Xue, Wang, Xianjun, Chen, Yi, Yang. Spatiotemporal control of gene expression by a light-switchable transgene system.[J]. Nature Methods, 2012, 9(3):266-9.
3.Stanley S A, Sauer J, Kane R S, et al. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles.[J]. Nature Medicine, 2015, 21(1):92-98.
4.Stanley S A, Gagner J E, Shadi D, et al. Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice.[J]. Science, 2012, 336(6081):604-.
5.Jean-Paul, Fortin, Claire, Wilhelm, Jacques, Servais, et al. Size-Sorted Anionic Iron Oxide Nanomagnets as Colloidal Mediators for Magnetic Hyperthermia[J]. J.am.chem.soc, 2007, 129(9):2628-2635.
6.Bou-Abdallah F, Yang H, Awomolo A, et al. Functionality of the Three-Site Ferroxidase Center of Escherichia coli Bacterial Ferritin (EcFtnA)[J]. Biochemistry, 2013, 53(3):483-495.
7.https://2008.igem.org/Team:TUDelft
8.Liang R, Liu X, Liu J, et al. A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.[J]. Journal of Microbiological Methods, 2007, 68(3):497-506.
9.Cai Y, Cao C, He X, et al. Enhanced magnetic resonance imaging and staining of cancer cells using ferrimagnetic H-ferritin nanoparticles with increasing core size.[J]. International Journal of Nanomedicine, 2015, 10(default):2619-34.
