Difference between revisions of "Team:NAWI Graz/Design"

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<b>Fig. 2:</b> The figure shows the molecular background to the acid-inducible promoter
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<b>Fig. 3:</b> The figure shows the molecular construction of the alkaline-inducible promoter
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             <a class="intralink" href="https://2017.igem.org/Team:NAWI_Graz/pHPlasmid#alx">Full description</a>
 
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<b>Fig. 4:</b> Bioreactor design. The basic construction of our reactor corresponds to standard bioreactor setup. Additionally it contains a variable measurement unit which can be adapted to different parameters.
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Revision as of 16:10, 1 November 2017

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 the different sections in "Parts").

The image cannot be displayed
Fig. 1: Overview of the whole system: a Thymio II robot sends its sensor values to the control server, who translates those values into environmental stimuli for the bacteria (in an interaction module (IM), shown here are the heat shock module and the pH-controller module). These stimuli trigger the expression of fluorescent proteins, which are detected in the fluorescence measurement chamber. If the fluorescence is strong enough, the server will tell the robot to change its behaviour.

The Bacteria

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

Full description
The heat shock promoter ibpA is controlled by the transcription factor σ 32. In principle, the exposure to high temperatures leads to an increase of σ 32, 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

Full description

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.

Fig. 2: The figure shows the molecular background to the acid-inducible promoter

"alx" - Alkaline-induced Roboswitch

Fig. 3: The figure shows the molecular construction of the alkaline-inducible promoter
Full description

Regulation of translation is 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.


The Bioreactor

Full description

Our system consist of several modules, we differentiate them in 3 layers seen in figure 3. 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 D 600 measure unit and the other one can be changed is a variable component (see Interaction Modules (IMs) and Fluorescence Measurement Chamber (FMC)). 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 D 600 value the D 600 measure unit also transports medium containing cell mass to the waste to regulate the D 600 value if it is too high.

Fig. 4: Bioreactor design. The basic construction of our reactor corresponds to standard bioreactor setup. Additionally it contains a variable measurement unit which can be adapted to different parameters.

Interaction Modules (IMs)

Full description

An IM receives bacterial suspension and exposes it to a stimulus, whose quantity is determined by the robot’s sensor readings. We have designed and built two IMs, one for heating up the suspension and a second one to set its pH (both ways). The “Variable Measuring unit” shown in Fig. 3 consists of at least one IM and an Fluorescence Measurement Chamber (FMC). The modularity of our design allows us to replace one IM for another (or even employ both) while keeping the rest of the setup intact.

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.
Fig. 5:

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.
Fig. 6: