DESIGN

The bacteria-robot interface is realized as a highly modular feedback system.

A mobile robot’s sensor values alter the environment of E. coli strains that were designed to respond to these changes with increased expression of fluorescent proteins. In turn, this fluorescence is measured and its quantities are translated into robot behaviour.

For how it all played out, see [[Results]], but let’s first have a glimpse at the modules comprising the feedback loop (for a full description see [[Parts]]):

The Bacteria

([[Parts#Bacteria|‘’full description’’]])

Our cultures have one goal: they should react to changes in their environment by expressing fluorescent proteins. Therefore, we introduced three promoters, two sensing the pH and one sensing the temperature of the culture media. Activation of one promoter leads to transcription and translation of a fluorescence protein.

"ibpA" - Heat Shock Promoter

The heat shock promoter ibpA is controlled by the transcription factor σ32. In principle, the exposure to high temperatures leads to an increase of σ32, which subsequently enables heat shock promoters to be recognized by the RNA polymerase. The promoter exhibits a high induction rate and high levels of expression. In our experiment, the ibpA promoter controls the expression of GFP.

"asr" - Acid Inducible Promoter

Promoter activity is controlled by the RstAB System detecting the pH and the PhoRB System activated when inorganic phosphate is rare. Thus, expression only works in low phosphate media (LPM). When grown in LPM and activated by a switch of pH to 5,5 the promoter becomes active and mCardinal is expressed. To enhance expression an extra ribosome binding site (RBS) was inserted between the promoter and mCardinal.

"alx" - Alkaline-induced Roboswitch

Regulation of translation ins managed by a pH sensitive riboswitch. The riboswitch itself, a mRNA part 5’ of the RNA coding for our green fluorescence mNeonGreen is regulated by a constitutive promoter.Hence regulation of mNeonGreen translation is managed by the riboswitch in subjected to pH. When pH is neutral the structure of the riboswitch prevents the ribosome from binding to the RBS. When pH rises, structure of the mRNA changes and allows the ribosome to bind the RBS and therefore translation can start. binding site (RBS) was inserted between the promoter and mCardinal.

The Bioreactor

([[Parts#Bioreactor|‘’full description’’]])

Our system consist of several modules, we differentiate them in 3 layers seen in figure XXX. The layer on top is the INPUT layer, it is a steady source of medium or variable liquid, which can be changed and is a key component for its variability. The ANALYSE and MAINTAIN layer consists of two elements and the reactor important to maintain our culture and two separate measuring units, one of which is the OD600 measure unit and the other one can be changed is a variable component (see [[Design#Interaction Modules (IMs)|IMs]] and [[Design#Fluorescence Measurement Chamber (FMC)|FMC]] below). Both the variable liquid and the variable measure unit gives us the freedom to change our system for different projects. In the OUTPUT layer we collect the waste from our measure units. Further to obtain a steady OD600 value the OD600 measure unit also transports medium containing cell mass to the waste to regulate the OD600 value if it is too high.

Interaction Modules (IMs)

([[Parts#InteractionModules|‘’full description’’]])

Our system consist of several modules, we differentiate them in 3 layers seen in figure XXX. The layer on top is the INPUT layer, it is a steady source of medium or variable liquid, which can be changed and is a key component for its variability. The ANALYSE and MAINTAIN layer consists of two elements and the reactor important to maintain our culture and two separate measuring units, one of which is the OD600 measure unit and the other one can be changed is a variable component (see [[Design#Interaction Modules (IMs)|IMs]] and [[Design#Fluorescence Measurement Chamber (FMC)|FMC]] below). Both the variable liquid and the variable measure unit gives us the freedom to change our system for different projects. In the OUTPUT layer we collect the waste from our measure units. Further to obtain a steady OD600 value the OD600 measure unit also transports medium containing cell mass to the waste to regulate the OD600 value if it is too high.

Temperature IM

In this module, tubes carrying the bacterial suspension are coiled between a Peltier-element and a styrofoam block allowing for quick heating of the suspension.

pH IM

This module consists of two lab bottles, containing acid and base solution. Two peristaltic pumps are controlled according to the sensor values of the robot, to specifically set the pH value of the reactor medium.

Fluorescence Measurement Chamber (FMC)

([[Parts#Fluorescence Measurement Chamber|‘’full description’’]])

A 3D printed case housing a cuvette (with in- and outflow tubes), LEDs to excite the fluorescent proteins, optical filters and a camera-module. We look into the raw RGB data in a region of interest to detect bacterial fluorescence. The camera is connected to and controlled by the server ([[Design#Server|see below]]).

Server

([[Parts#Server|‘’full description’’]])

A Raspberry Pi [explain? footnote?] that coordinates the behavior of all the components of the system as well as offering an interface to users. It maintains a steady-state in the bioreactor, pumps bacterial suspension from the reactor into the other modules (the [[Design#Interaction Modules (IMs)|IM]] and [[Design#Fluorescence Measurement Chamber (FMC)|FMC]]), controls the behavior of those and communicates with the robot by receiving sensor readings and sending commands.

Robot

([[Parts#Robot|‘’full description’’]])

We hooked up a Raspberry Pi [explain? footnote?] to a Thymio II educational robot [explain? footnote?] in order to enable it to communicate with a server on the internet. The robot moves around in a maze and streams its various sensor readings to the server, while in return receiving commands from the server (according to bacterial fluorescence) to change its behavior.

Arena

([[Parts#Arena|‘’full description’’]])

A collection of wooden boards and obstacles that can be used to set up a great variety of environments for the robot, some simple, others more challenging.

Design

Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.

This page is different to the "Applied Design Award" page. Please see the Applied Design page for more information on how to compete for that award.

What should this page contain?

Explanation of the engineering principles your team used in your design
Discussion of the design iterations your team went through
Experimental plan to test your designs

Team:NAWI Graz/Design