"
Team:Braunschweig/Project/Appoach
From 2013.igem.org
| Line 7: | Line 7: | ||
#contents {width: 850px; margin-left: 50px; margin-top: 100px; margin-right: 60px; clear:both | #contents {width: 850px; margin-left: 50px; margin-top: 100px; margin-right: 60px; clear:both | ||
} | } | ||
| - | + | #sponsors { | |
| + | clear: both; | ||
| + | margin-left: 45px; | ||
| + | margin-right: 65px; | ||
| + | margin-top: 100px; | ||
| + | } | ||
</style> | </style> | ||
| Line 13: | Line 18: | ||
<strong>Project – Approach</strong></p> | <strong>Project – Approach</strong></p> | ||
<img alt="linie rot 8pix hoch" src="https://static.igem.org/mediawiki/2013/0/07/Team_Braunschweig_Red_line.jpg" width="850" height="1" /></p> | <img alt="linie rot 8pix hoch" src="https://static.igem.org/mediawiki/2013/0/07/Team_Braunschweig_Red_line.jpg" width="850" height="1" /></p> | ||
| - | + | <p>Approach | |
| + | |||
| + | 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 overgrow 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.<br> | ||
| + | In order to creat a dependency among the strains present in the culture we decided to base our project upon three different Quorum Sensing systems from Pseudomonas and Vibrio fischeri (Las, Rhl and Lux system) and three different E. coli strains. The communication among the strains works via different autoinducers 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-gen (ampR) which is located downstream of an inducible promotor (plas, prhl, plux) and is only induced in the presence of a specific constitutively expressed transcrption activator (lasR, rhlR, luxR) together with the corresponding autoinducer. Therefore the strains rely on the presents of the others in the culure broth.<br> | ||
| + | In order to analyse the culture composition the idea of the first cloning strategy was to include different fluorescent markers and using FACS in order to determine the number of cells from each strain. However, with the equipment available we cannot detect three different fluorescent markers at the same time due to their excitation and emmision spectra. Hence we modified our strategy to use chromoproteins designed by iGEM Uppsala 2011 instead, since most of these colors fluoresce and all are visible to naked eye. The respective cloning strategy is shown below. By producing the chromoproteins the cells in the culture broth became very sticky which might correlate with the chromoproteins expression thus we were not able to analyse single cells in the FACS. However we are still able to analyse the composition by naked eye.<br> | ||
| + | Having severe trouble with the cloning of the Lux-system for a long time we are forced to switch our system to two strains depending on the Las and Rhl system.<br> | ||
| + | All experiments with the finale gene constructs are carried out with only these two strains. In order to verify the function of every part 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 continous culuture. | ||
| + | </p><p><img alt="Cloning strategy" src="https://static.igem.org/mediawiki/2013/7/75/Braunschweig_Lab_Journal_May_27.png" width="850" vspace="0" hspace="20"/></p> | ||
</div> | </div> | ||
| Line 20: | Line 32: | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<div id="sponsors"> | <div id="sponsors"> | ||
<strong>Our sponsors</strong></p> | <strong>Our sponsors</strong></p> | ||
Revision as of 21:29, 17 September 2013
Approach
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 overgrow 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 creat a dependency among the strains present in the culture we decided to base our project upon three different Quorum Sensing systems from Pseudomonas and Vibrio fischeri (Las, Rhl and Lux system) and three different E. coli strains. The communication among the strains works via different autoinducers 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-gen (ampR) which is located downstream of an inducible promotor (plas, prhl, plux) and is only induced in the presence of a specific constitutively expressed transcrption activator (lasR, rhlR, luxR) together with the corresponding autoinducer. Therefore the strains rely on the presents of the others in the culure broth.
In order to analyse the culture composition the idea of the first cloning strategy was to include different fluorescent markers and using FACS in order to determine the number of cells from each strain. However, with the equipment available we cannot detect three different fluorescent markers at the same time due to their excitation and emmision spectra. Hence we modified our strategy to use chromoproteins designed by iGEM Uppsala 2011 instead, since most of these colors fluoresce and all are visible to naked eye. The respective cloning strategy is shown below. By producing the chromoproteins the cells in the culture broth became very sticky which might correlate with the chromoproteins expression thus we were not able to analyse single cells in the FACS. However we are still able to analyse the composition by naked eye.
Having severe trouble with the cloning of the Lux-system for a long time we are forced to switch our system to two strains depending on the Las and Rhl system.
All experiments with the finale gene constructs are carried out with only these two strains. In order to verify the function of every part 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 continous culuture.
