Team:Pittsburgh/Human Practices
Human Practices
Input by Experts
“Magnetogenetics” is a relatively new and developing research tool. In order to ensure that our project design is viable, feasible, and responsible for the world, we contacted several experts with specializations in three main aspects of our project design: building the AMF emitting device, selecting and attaching magnetic nanoparticles, and selecting heat-sensitive outputs. By conferring honestly with these experts about our proposed project plan and progress, our intention was to optimize the main features of our project into the most viable design for our world possible. With these responsible and feasible advisements and adjustments throughout our project development, it is our hope that our project Bluetooth Bacteria will one day be able to serve our community in capacities featuring medicinal therapeutics, scientific innovation, and industrial implementation.
| Expert | Institution | Expertise |
|---|---|---|
| Dr. Mikhail Shapiro, PhD. | California Institute of Technology (Caltech) | Temp sensitive TF |
| Dr. Dan Piraner, PhD. | University of California, San Francisco (UCSF) | Temp sensitive TF |
| Dr. Polina Anikeeva, PhD. | California Institute of Technology (Caltech) | Magnetic hyperthermia |
| Dr. Siyuan Rao, PhD. | Massachusetts Institute of Technology (MIT) | Magnetic nanoparticle |
| Dr. Michael Christiansen, PhD. | ETH Zurich | AMF device |
| Dr. Warren Ruder, PhD. | University of Pittsburgh | Magnetic nanoparticles |
AMF Device to stimulate Magnetic Nanoparticles
Our initial understanding of how to stimulate magnetic nanoparticles (MNP) was limited. Radio waves (RF) were reported to heat up superparamagnetic nanoparticles (Stanley et al, 2012), so our first idea was to use an ESG signal generator emit low-frequency radio-waves in the range of 100 kHz to 6 GHz to heat MNP. For advice on our design, we contacted Dr. Polina Anikeeva, an expert in designing magnetic devices for the treatment of psychiatric disorders. She explained a common misconception regarding the nomenclature of radio-waves and alternative magnetic frequencies (AMF), directing us away from the utilization of radio-waves, and instead encouraging us to stimulate our nanoparticles with AMF. To stimulate the magnetic particles, Dr. Anikeeva informed us an alternating magnetic field (AMF) with radio frequencies ranging between 15 kHz to 2.5 MHz is generally used (Christiansen et al. 2017). We realized we could not use the ESG signal generator to emit RF as we originally planned and needed to shift our focus to building an AMF emitting device for MNF stimulation instead. For help in designing the AMF device, Dr. Anikeeva directed us to Dr. Michael Christiansen.
We met with Dr. Michael Christiansen for advice on building the AMF emitting-device, as our knowledge in the area was limited. After discussing our project plan with Dr. Christiansen, we decided to model our AMF-emitting device based on the E-gap described in Christiansen et al (2017). Dr. Christiansen provided information on what equipment and materials we will need such as an amplifier, oscilloscope, function generator, capacitor, transformer, and a shunt resistor. He highlighted important considerations for measurement, including difficulties involved with discerning the amount of heat produced from the magnetic nanoparticles and lost from the device. He also suggested building a fan or using a water bath to cool the device.
Another major aspect of our design is to control and maintain the temperature of the bacterium in order to control the heat-sensitive gene output. As AMF stimulation increases, the heat generated by MNPs increases linearly (Shah et al. 2015). In order to maintain our desired temperature to observe this kind of trend, Dr. Christiansen suggested we will have to periodically turn the device on and off.
Selecting and Attaching Magnetic Nanoparticles
Dr. Anikeeva also suggested we contact Dr. Siyuan Rao for advice on working with MNPs. Unsure of which brand or type of MNP to purchase for our experimental needs, Dr. Rao recommended we purchase our MNP commercially. Specifically, she recommended we buy either the magnetic beads or iron oxide nanoparticles from the company Oceantech. She also explained that the choice of MNP size is determined by Specific Loss of Power (SLP). The size of the MNP’s suggested to us was 25 nm in size, which yielded the highest heat dissipation value (Chen et al. 2017; Christiansen et al. 2017).
Dr. Rao suggested that specific localized heating is very difficult to accomplish since she had some experience with it while trying to induce localized heating in mammalian cells. After discussing with her, we shifted our focus from trying to only heat up the area around the cell membrane to creating bulk heating in the bacterial solution. The surface chemistry would then serve to bring the heating closer to the cells as well as create more homogenous heating. She explained that the type of coating on the magnetic nanoparticle (e.g. carboxy vs amine) does not change their overall response to AMF. With Dr. Rao’s abundance of helpful advice, we were able to begin deciding what MNPs we wanted to purchase based on size, and the type of surface chemistry we were looking to achieve to best fit our binding needs.
To receive more guidance on the utilization of magnetic nanoparticles, as well as how to attach them to bacterial membranes, we contacted Dr. Warren Ruder. He suggested that instead of using the streptavidin-biotin and chitosan attachments, we should use antibodies, which have the capacity to bind to specific proteins on the bacterial surface. Dr. Ruder hypothesized that one of the reasons why our streptavidin plasmid transformations experiments were unsuccessful was due to streptavidin binding to the biotin inside the cell. This accidental binding would decrease cellular function and cause the cells to grow slower. In addition, the streptavidin that we express may already be bound to biotin by the time it reaches the surface. Dr. Ruder also offered advice on how to work with the magnetic nanoparticles from his own experience. For instance, we should work with nanoparticles suspended in PBS rather than LB, since LB contains proteins that can possibly interfere with the coated nanoparticles. He also suggested having the bacterial solution shaken while exposing it to the AMF to prevent the nanoparticles from precipitating.
Heat-sensitive output
To induce GFP expression, we required a part to respond to the cytoplasmic change in temperature. Initially, we were considering using heat-sensitive dimers, heat-shock promoters, or RNA thermosensors to regulate gene output. In Piraner et al. (2017), TlpA and TcI heat-sensitive dimers were designed as a thermal bio-switch, which seemed very promising. We met with Dr. Dan Piraner to receive advice on working with these heat-sensitive dimers.
Our original plan was to conduct our experimentation with all the heat-sensitive promoters, expecting all to induce gene expression in E. coli similarly. Dr. Piraner clarified that misconception and indicated that TlpA and TcI would be best to use as a thermal bio-switch compared to using heat-shock promoters due to their higher fold induction, orthogonality, and tunability. He also provided advice regarding how to work with these heat-sensitive plasmids. He provided us with a general protocol for measuring expression, including the OD the cells should grow to. In addition to offering a comprehensive protocol, he included specialized notes that he acquired from experience in running the experiment. He noted that TlpA was “leaky” and should be grown at 30 °C, and to run the temperature-induction experiments, we should set up multiple PCR blocks at different temperature points, letting the cells incubate for around 6-12 hours.