Team:Oxford/biosensor construction1

From 2014.igem.org

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<li>The two plasmids are partitioned during cell division by different systems, thus an equal proportion of each plasmid is maintained in each new daughter cell. </li><br> <li>Different antibiotic resistances will allow us to select for cells that have taken up both plasmids by application of both antibiotics.</li><br> <li>The replication origins compatible with E.coli and pseudomonas strains.</li><br> <li>We have used two plasmids so that we can test each part in isolation before transforming them both into the same cell.</li>
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<h1>Predicting the sfGFP fluorescence</h1>
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<h1>Introduction</h1>
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To allow us to characterize the second half of the genetic circuit, we needed to be able to predict the difference in response. To do this, we constructed models by cascading the differential equations according to the respective circuit structures thereby producing two different potential system responses.
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To achieve this, we constructed simplified equivalent circuits that were linked by two potential activation-repression relationships.
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It is important to understand that these simplified equivalent circuits will not give the correct mCherry response but they will give the correct GFP response after correct parameterisation.
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We then set up the differential equations necessary to solve this problem in Matlab. The method and results are as detailed below:
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<a href="https://static.igem.org/mediawiki/2014/b/be/Oxford_Equations_explained.png"><img src="https://static.igem.org/mediawiki/2014/4/41/Oxford_equations.png" style="float:left;position:relative; width:47%;" /></a>
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<a href="https://2014.igem.org/Team:Oxford/biosensor_modelling_approximations"><img src="https://static.igem.org/mediawiki/2014/0/02/Oxford_approximations.png" style="float:right;position:relative; width:47%; margin-left:75%, margin-top:3%" /></a>
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<h1>Conclusion</h1>
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The bottom graphs illustrate the predicted response of each system to a simultaneous step input of both DCM and ATC. As you can see, there is little difference in the predicted steady-state value of the fluorescence, however, providing the basal transcription rate of GFP is relatively low, there should be a clear difference in the level of fluorescence before either of these inputs are added. This very easily identifiable difference between the two systems will enable us to characterize the genetic circuit present in our particular system.
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<h1>Calculating the parameters</h1>
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Calculating the many parameters for this system will be undoubtedly challenging.
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<a href="https://2014.igem.org/Team:Oxford/calculating_parameters">How are we calculating the parameters?</a>
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<a href="https://2014.igem.org/Team:Oxford/biosensor_characterisation#hide6">Go to the data section where we calculated parameters for this part of the circuit.
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Revision as of 21:06, 11 October 2014


Construction


Introduction: how we constructed our biosensor

In order to be able to use our model and to determine whether DcmR acts as a repressor or activator in the presence of DCM we designed and constructed the following two plasmid system. We primarily used Gibson assembly methods and source most of the necessary DNA from gblocks(synthesised oligonucleotides) we had designed based in the sequenced genome of Methylobacterium DM4. This system will also form the DCM biosensor and will be integrated with an electronic circuit to complement this genetic one:

pOXON-2-dcmR-mCherry
pOXON-2-dcmR-mCherry
Oxford iGEM 2014
pSRK-Gm-pdcmAsfGFP
pSRK-Gm-pdcmAsfGFP
Oxford iGEM 2014
Why these two plasmid backbones?
Why these two plasmid backbones?
  • The two plasmids are partitioned during cell division by different systems, thus an equal proportion of each plasmid is maintained in each new daughter cell.

  • Different antibiotic resistances will allow us to select for cells that have taken up both plasmids by application of both antibiotics.

  • The replication origins compatible with E.coli and pseudomonas strains.

  • We have used two plasmids so that we can test each part in isolation before transforming them both into the same cell.
    • How were the constructs made?
    How were the constructs made?

    Wetlab data showing response in level of mCHERRY expressed with different concs of ATC

    data analysis



    Oxford iGEM 2014

    Retrieved from "http://2014.igem.org/Team:Oxford/biosensor_construction1"