Team:SUSTech Shenzhen/Design
Design
Project
Introduction
In biology science, many complex experiment result are not repeatable because of high dimension of variables. Too many uncontrollable variables make the output unpredictable. For example, gene expression is a complex and stochastic process involving numerous component and reaction steps.[1] With continue development in synthesis biology, many new ways is come up with, including chemical genetics [2], optogenetics [3, 4]. Chemical genetics use small molecular drugs such as aTc to induce the gene expression. Optogenetics use light as input signal to induce gene expression. However, they all have their own limitations or drawbacks. Chemical genetics lack spatial selectivity or specificity, beside, because the drugs is not stable in vivo, it can be degraded and the concentration of small chemical in cell may not the same as in extracellular. The side effect of optogenetics is from laser-induced heating, abnormal ions distribution caused by overexpressed pumps or channels, and/or undesired network homeostasis can make experimental interpretation very difficult [5].
Living organisms needs to deal with different environments, sound is a kind of signal of the changing environment. As founded in human’s ears, mechanosensitive (MS) channels are taking responsibility for the hair cells to response to sound. [6] For other systems to sense the sound, in the plant, cell membrane-associated proteins like MS channels are suggested related to this ability. [7] Plants are founded to response to sound of specific frequency and intensity. [8] We wants to find out if this can be related to MS channels. There are some researches have been done in discover the relationship between MS channels and sound, but experiment have been done on cell level, most of them are employing ultrasound. [9] .
In this study, we invented a new technique named as audiogenetics, which can precisely and nontoxicly regulate the gene expression in cells. The activation of gene expression is executed through a mechanosensitive channel TRPC5 [11-13] or piezo channel[14, 15], they can sense the mechanical force and open the gate to selectively permeate calcium, also, the influx of calcium can promote the activation of TRPC5 channel and more calcium will influx into cytosolic [4]. We use a fluorescent indicator, R-GECO, to quantify the intracellular calcium, it derive from GCaMP. After it bind with calcium, it can emit fluorescent with about 600 nm wavelength [16]. Calcium in cytosolic can regulate a series of phosphorylation, in the downstream of calcium signal we use the specific promoter (PNFAT) and the transgene expression (GFP) as the output signal to quantitatively analysis the regulatory ability of audiogenetics [4]. We will test this system in CHO-K1 cell line, because CHO-K1 can be culture suspendingly, which is convenient for us to active the TRPC5 or piezo with sound wave. In addition, to quantitatively examine the sensibility of those MS channel to mechanical stress, we firstly active those MS channel by microfluidics [17] and hypoosmolarity [18] and observe the calcium influx. Then, we use sound of different combination of frequent and intensity to active those MS channel, and we made it by using some commercial sound generator such as buzzer, balanced armature, horn, ultrasound and atomizer. The successful combination of audiogenetics stimulation and genetic targeting will reshape the landscape of currently available gene regulation. This novel technology will open a new door to invasive, audio control of gene expression, neuronal activities and other biological process in cells or organisms.
Measurement
Test of R-GECO
GCaMPs consist of a circularly permuted enhanced green fluorescent protein (such as EGFP), which is flanked on one side by the calcium-binding protein calmodulin and on the other side by the calmodulin-binding peptide M13. In the presence of calcium, calmodulin-M13 interactions elicit conformational changes in the fluorophore environment that lead to an increase in the emitted fluorescence. (Fig. 1a) We can use histamine to induce calcium influx in HeLa cell to test R-GECO calcium indicator [19], (Fig. 1b) however, there is no H1 receptor in CHO-K1 cell membrane.
