A: From the Internet to Bio-Electronic Gene Regulatory Networks

Here the Internet is connected to living organisms through an Arduino based device that can take AC or DC signals as inputs, gates them through 6 relays and dispatches them individually to each of the 5 pads of the Electro-Micro-Slide or to the each individual wells of the High Throughput Electro Actuator (HTEA). It is controlled by the Electro Planner, a device also used in the screening experiment and most of the membrane-potential-related preliminary work. An event happening on the Internet, here a tweet, is capable of transmitting a command to the material environment of bacterial cells.

B: Electrical signal transduction

To transduce electrical signals to biochemical processing systems in gene regulatory networks and perform useful computations, we engineered bacterial population with a 3-plasmid system:

An input system: translating electrical signal into chemical ones.
A processor circuit: transforming the input signal through common synthetic biology operations.
An output system: translating the results of the previous computation into computer readable signals.

We first characterised each part individually, and then assembled and assessed the entire system. (For more information, visit our Engineering page)

Input Systems

Redox sensing system: - pSoxS - dpB.002 experiment

Our Input plasmid is composed of a Transcriptional Unit consisting of an electrically induced promoter placed upstream of the cinR gene. This gene encodes for the CinR transcription factor that activates the pCin promoter in the presence of OCH14 (the chemical inducer of the system).

We tested and characterised multiple Electro-Genetic promoters found in literature, which rely on the Redox Sensing mechanisms of E. coli.

PsoxS is part of the redox sensing mechanism of E. coli. It is activated by the SoxR transcription factor in its oxidised state. As shown in recent work in electro-genetics [1], the addition of oxydized pyocyanin to the media induces the pSoxS expression via SoxR. Reducing pyocyanin through applied constant voltage allows for electro-genetic control of PsoxS expression.

We cloned the dpB.002 (Bba_K4216027) into the V35 vector and characterised the variation of mScarlet expression as a function of pyocyanin concentration.

dpB.002

High throughput screening of electrically inducible promoters - Experiment results

To increase the range of available bio-digital interfaces, we conducted a large scale screening experiment of E. coli's' repertoire of promoters in search for new electrically-responsive promoters. We used the High-Throughput Electro Actuator (HTEA), a custom-made array of electrodes that can be used to dispatch signals from the AC Dispatcher (ACD) to each of the individual wells of 96-well plates. We exposed the E. coli Promoter Collection [3] to a variety of electrical signals.

Fold change in the fluorescence signal of the promoters of the collection was used to identify electrically-responsive promoters. This methods allowed us to identify and characterised 19 promoters (8 induced and 11 repressed) responding to specific electrical signals.

Processor System - dpB.010 Experiment

We cloned the dpB.010 (Bba_K4216035) [-Cin-mScarlet] into the V35 Vectors.

In accordance with our designs (see Engineering), biomolecular signals produced by the Input plasmid are passed down to the transduction pathway. To prototype components individually (without the upstream signal in another plasmid) , we transformed the resulting constructs into the Wild Marionnette strain[4].

The Marionettes are engineered E. coli strains which constitutively express 12 Transcription factors (TF) modulating the activity of their cognate promoters. Our 3-plasmid system rely on 2 of these 12 induction systems to pass the signal. Working with the Marionette Strain therefore allows us to bypass the action of upstream signals (as they are constituvely expressed in these strains).

In this construct, an mScarlet is placed under the control of the pCin promoter (one of the marionette promoter regulated by its cognate TF, CinR and its inducer OHC14).

We observed the variation of Fluorescence of mScarlett as a function of inducer concentration and validated the functioning of the Processor system on its own.

dpB.010

Output system - Induced Lysis Experiment

We cloned the dpB.014 (Bba_K4216039) pLux78B - φX174 (Lysis)[] into the V35 vector and transformed this construct in the Marionette strain [4].

Here, we placed the φX174 lysis gene under the control of the pLux78B promoter (one of the marionette promoters regulated by its cognate TF LuxR and its chemical inducer OC6).

We observed the variation of final optical density (OD600) reached as a function of inducer concentration and validated the functioning of the Output system on its own. The range of final optical density values are proportional to the variation of gene expression. This demonstrates that controlling lysis can be electronically retrievable data.

dpB.014

C: The 3 Plasmid system

Once the components of our system tested and validated individually, we conducted dose response experiment of our 3-plasmid system.

We transformed an E. coli MG1655 with the following 3 constructs assembled in our modified compatible pDuet vectors:

Input Plasmid: dpB.003 (Bba_K4216028) [pSoxS-CinR] - pACYDuet_Bettencourt
Processor Plasmid: dpB.011 (Bba_K4216036) [pCin - LuxRQ] - pCDF_Bettencourt
Output Plasmid: dpB.013 Bba_K4216038) [pLuxB - mScarlett] - pCOLADuet_Bettencourt

As shown in [1], addition of pyocyanin to the growth media induce the expression of pSoxS. We grew our colonies transformed with the 3 plasmids at different concentration of pyocyanin and measured the fluorescence of our output system.

Our result show that a signal transduced in the input plasmid modulates the quantity of fluorescent protein produced from the output plasmid.

This experiment validates the ability of our 3-plasmid system to carry chemical signals throughout our system.

Signal modulation Experiment

D: From biochemical information to digital bits

To translate the information of the output system (the final OD reached after growth), we developed a way to electronically monitor cell density. As shown in [5], a growing population of bacteria capture ions from the medium and therefore decrease the medium electrical conductivity. We used our adapted IO Rodeo Potentiostat to measure of electrical resistance as a mesure of OD.

Electrical Resistance as a measure of cell density Experiments

Interfacing Biology to Existing Digital Systems

Because of the electronic nature of our Input/Output system, essentially black-boxing biological processes into electronics, interfacing with varied electronic system is achievable through classical Electronic-Electronic interfaces. To explore the scope of possible future interaction enabled by our toolkit, we seek applications through varied fields and approach problems in Bio-production, Robotics and sensing, Music production and new media Arts.

Go to our Human Practices

In summary

We showed that our toolkit was capable of sending electrical signals in response to events occurring in the Internet (Part A).
We showed that these electrical signals were sufficient to control gene expression and convert electrical information into biochemical ones (Part B).
We showed that our system is capable conveying these molecular information throughout a modular signal processing pipeline composed of 3 plasmids (Part C).
We showed that variation of expression in our output system was an electronically retrievable information (Part D).

Taken together these results represent a strong proof of concept of the integration of synthetic biology in networked electronic systems through bio-digital interfaces.

Proof of Concept

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