Project – Approach

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The aim of our project was to engineer a Synthetic Microbial Consortium solely based on standard parts available from the 2013 registry. Microbial consortia are used in lots of biotechnological processes for example bioremidation of contaminated soil and waste water treatment. These consortia, however, grow completely uncontrolled. In some cases it would be favorable for the process if the microorganisms would be present in a definded ratio. In fermentation processes defined mixed culutres cannot be employed to carry out synthesis of a product or bioconversion since one strain will usually outgrow the others causing them to vanish from the culture broth. Thus our aim was to implement a system for controlled growth of mixed cultures to overcome this problem by engineering a synergetic system.
In order to create a dependency among the strains present in the culture we decided to base our project upon three different Quorum Sensing systems from Pseudomonas aeruginosa and Vibrio fischeri (Las, Rhl and Lux system) and three different E. coli strains. The communication among the strains works via different so called autoinducers, often homoserinelactones, produced by the QS-systems synthetase (LasI, RhlI, LuxI) which are secreted into the surrounding media and taken up by other cells. Each strain carries an antibiotic resistance-gene (ampR) which is located downstream of an inducible promotor (Plas, Prhl, Plux) and is only induced in the presence of a specific constitutively expressed transcription activator (LasR, RhlR, LuxR) together with the corresponding autoinducer. Therefore the strains rely on the presence of the others in the culture broth.
In order to analyse the culture composition by flow cytometry the idea of the first cloning strategy was to include different fluorescence markers. However, with the equipment available we were not able detect three different fluorescence markers at the same time due to their excitation and emmission spectra. Hence, we modified our strategy to use chromoproteins designed by the iGEM Uppsala team 2011 instead, since most of these colors fluoresce and are all visible to the naked eye. The respective cloning strategy is shown below. We were not able to analyse single cells via flow cytometry as cells expressing the chromoproteins built clusters. However, by plating the cultures and counting colonies we were still able to analyse the composition.
Having severe trouble with the cloning of the Lux-system for a long time we were forced to change our system to two strains depending on the Las and Rhl system.
All experiments with the final gene constructs are carried out with only these two strains. In order to verify the function of each operational unit of our constructed devices we run tests on promotor induction and leakyness, synthesis of autoinducer molecules using reporter strains and dependency of the strains in batch as well as in continuous culture.

Cloning strategy

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