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| - | <img src="https://static.igem.org/mediawiki/2014/6/6f/Oxford_Characterisation_question.png" style="float:right;position:relative; width:42%;" /> | + | <img src="https://static.igem.org/mediawiki/2014/b/b6/Oxford_pOXON-2-dcmR-mCherry_text.png" style="float:left;position:relative; width:100%;" /> |
| - | <h1>DcmR and regulation of <font style="font-style: italic;">dcmA</font> expression</h1>
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| - | Mutants with <font style="font-style: italic;">dcmA</font> and the intergenic region but without complete <font style="font-style: italic;">dcmR</font> express DcmA constitutively. Re-integration of <font style="font-style: italic;">dcmR</font> restores regulation of <font style="font-style: italic;">dcmA</font> expression at the transcriptional level [1]. In addition, it has been shown that the region including <font style="font-style: italic;">dcmR</font>, the intergenic region and <font style="font-style: italic;">dcmA</font> is sufficient to confer a DCM dependent response in genetically engineered Methylobacterium extorquens DM4 [2]. <br><br>
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| - | <h1>DcmR and DNA-binding</h1>
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| - | DcmR is thought to be a DNA binding protein as structure predicting software indicates that there is a helix-turn-helix domain at the N-terminal of the protein. Since the region between the two promoters for <font style="font-style: italic;">dcmR</font> and <font style="font-style: italic;">dcmA</font> can be deleted without any effect on regulation it has been suggested that DcmR does not to a secondary regulatory site in between the genes but most likely acts directly on the <font style="font-style: italic;">dcmA</font> promoter itself [1]. In addition, regulated expression of <font style="font-style: italic;">dcmA</font> is not effected when the <font style="font-style: italic;">dcmR</font> and <font style="font-style: italic;">dcmA</font> transcriptional units are placed on separate replicons thereby suggesting that their topology is independent of the regulatory network. It is therefore suggested that DcmR binds the DNA in the intergenic region with the simplest model of its mode of action being as a trans-acting DNA-binding repressor; however this remains to be fully validated [1].<br><br>
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| - | We have therefore proceeded on the assumption that DcmR is directly influenced by the presence or absence of DCM and furthermore that we can use <font style="font-style: italic;">dcmR</font>, the intergenic region and <font style="font-style: italic;">dcmA</font> alone to characterise the regulatory network. <br><br>
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| - | [1] La Roche, S. D., and T. Leisinger. "Identification of <font style="font-style: italic;">dcmR</font>, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4." Journal of bacteriology 173.21 (1991): 6714-6721. <br>
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| - | [2] Lopes, N., et al “Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter” J Ind Microbiol Biotechnol (2012) 39:45–53
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| - | <h1>Characterising the DcmR - DCM - P_dcmA interaction</h1>
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| - | To find out whether the gene <font style="font-style: italic;">dcmR</font> acts as a repressor or an activator on the promoter of the <font style="font-style: italic;">dcmA</font> gene, we attempted to build the genetic circuit shown above on the right. Having <font style="font-style: italic;">dcmR</font> under inducible TetR expression should allow us to have very good control of the amount of DcmR present. On top of this, attaching the mCherry fluorescence tag will act as another confirmation to the amount of DcmR present.
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| - | We then extensively modelled the circuit to discover how the response of the system would differ if it was either of the two circuit systems. Click the modelling bubbles (pink) to find out exactly how we achieved this.
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| - | <h1>Predicting the mCherry fluorescence</h1> | + | <img src="https://static.igem.org/mediawiki/2014/1/12/Oxford_pSRK-Gm-pdcmAsfGFP_text.png" style="float:left;position:relative; width:100%;" /> |
| - | We simplified the first double repression by modelling it as an activation of <font style="font-style: italic;">dcmR</font> by ATC, albeit parameterised by different constants. This assumption is justified by the fact that we are able to precisely control the addition of ATC and measure the fluorescence of the mCherry.
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| - | We modelled this first step using both deterministic and stochastic models.
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| - | <div class="white_news_block2">
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| - | <h1>Biochemical equations</h1>
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| - | The biochemical equations that describe the behaviour of the top half of the genetic circuit are:
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| - | <img src="https://static.igem.org/mediawiki/2014/f/ff/Oxford_mCherry_circuit2.png" style="float:right;position:relative; width:40%;margin-left:30%;margin-right:30%;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/1/1d/Oxford_biochem_equations.png" style="float:right;position:relative; width:80%;margin-left:10%;margin-right:10%;" />
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| | Oxford iGEM 2014 | | Oxford iGEM 2014 |
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| - | <h1>Deterministic</h1>
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| - | <img src="https://static.igem.org/mediawiki/2014/2/2e/Oxford_DcmR_parameters.png" style="float:right;position:relative; height:8%; width:47%;" />
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| - | Deterministic models are very powerful tools for synthetic biology. They describe the behaviour of the bacteria at the population level and use Ordinary Differential Equations (ODEs) to relate each activation and repression. By constructing a cascade of differential equations one can build a realistic model of the average behaviour of the system.
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| - | The differential equation that descibes this first step of the system is:
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| - | <br><br><img src="https://static.igem.org/mediawiki/2014/e/ed/Oxford_DcmR_activation.png" style="float:left;position:relative; height:8%; width:47%;" />
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| - | <a href="https://static.igem.org/mediawiki/2014/b/be/Oxford_Equations_explained.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/41/Oxford_equations.png" style="float:left;position:relative; width:40%;" /></a>
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| - | Solving this ODE in Matlab <u>(with zero basal transcription rate)</u> predicts the following the response of the system:
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| - | This model works assuming that <u>sufficient TetR</u> is always present.
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| - | <img src="https://static.igem.org/mediawiki/2014/e/e0/Oxford_MCherry_circuit.png" style="float:left;position:relative; width:40%;" />
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| - | <img src="https://static.igem.org/mediawiki/2014/9/92/MCherry_graph.png" style="float:right;position:relative; width:60%;margin-bottom:3%;" />
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| - | Oxford iGEM 2014
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| - | While the analysis of this circuit is not critical to the successful outcome of this part of the project, it will provide us with very good practice of both obtaining fluorescence time series data and accurately fitting the data to the model. It will also help us develop our methods of predicting future system behaviour. This is because this system is already well documented in the literature and so we should be able to test our methods and responses against well documented results from labs across the world.
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| - | As you can clearly see from the graph, the model predicts a large fluorescence increase as the input is added. This is the what we expect from the actual system and is the best approximation that is obtainable before we get experimental data.
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| - | In the graph above, the model is set to have a basal transcription rate of zero. This is why there is a zero fluorescence response before the input has been added - this corresponds to the tetO promoter not being leaky at all. This basal rate will be calibrated alongside all of the other parameters in the model.
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| - | <h1>Stochastic Modelling</h1>
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| - | Stochastic modelling uses probability theory to predict the behaviour of a system. For our project, we used it to model the expression of GFP from bacteria.
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| - | We started with the Gillespie Algorithm, which considers the expression of GFP to be binary; a molecule of GFP is either produced or degraded. We modelled the probability of a molecule of GFP being created using the Michaelis-Menten model, incorporating a basal transcription rate. For the degradation, we assumed a simple proportional relationship; the more you have the more likely it is that a molecule degrades. The constant of proportionality will be a function of the intrinsic life time of the protein in the cell. Now at every increment in time we will not have a GFP reaction occurring, so before we decided what reaction occurs we had to work out if I a reaction occurred. We did this by writing an equation involving the probability of any reaction occurring with a random number generator. To work out which reaction occurred we compared the relative probability of a production to degradation, and used a random number to make a weighted choice.
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| - | We later changed this code so that a reaction occurred every time increment, but included a null reaction where no GFP was degraded or created. Although this made the code a lot more data heavy, it allowed for much easier calculation of the mean response of multiple realisations.
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| - | Stochastic modelling is useful because it can show us the stochastic effects which are often seen in real bacteria. By calculating the variation of the mean of multiple GFP producing bacteria, we can also work out the standard deviation. Then if we assume that the system varies with respect to the normal distribution, we can produce error bounds for the production of GFP. Such that we can say, 90% of the time we can expect the production of GFP from a single bacterium to be within these 2 curves. This could be useful for seeing if results are unexpected, or, if there are multiple outliers, that our model is incorrect. If we average more and more bacteria then the mean curve tend towards the deterministic response. This is to be expected as we are now looking at the system as a whole and fluctuations in the production from individual bacteria are averaged out. Sto for small Det for large
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| - | What is stochastic modelling? -- Yes
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| - | How is it useful? Ads/Dis -- Getting there
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| - | Tending to deterministic -- Yes
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| - | Modelling activator repressor --?
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| - | Parameter characterisation/Data matching -- Not yet
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| - | Matlab graphs – Not yet
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| - | Improve Gillespie algorithm bit
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| - | <li>Matlab graphs</li>
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| | <a href="#show3" class="show" id="show3"><div class="wetlab"> | | <a href="#show3" class="show" id="show3"><div class="wetlab"> |
| - | <h1white>pSRK-Gm-pdcmAsfGFP</h1white> | + | <h1white>Why these two plasmid backbones?</h1white> |
| | <img src="https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png" style="float:right;position:relative; width:2%;" /> | | <img src="https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png" style="float:right;position:relative; width:2%;" /> |
| | </div></a> | | </div></a> |
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| | <a href="#hide3" class="hide" id="hide3"><div class="wetlab"> | | <a href="#hide3" class="hide" id="hide3"><div class="wetlab"> |
| - | <h1white>pSRK-Gm-pdcmAsfGFP</h1white></div></a> | + | <h1white>Why these two plasmid backbones?</h1white></div></a> |
| | <div class="list"> | | <div class="list"> |
| | <div class="white_news_block2"> | | <div class="white_news_block2"> |
| - | <img src="https://static.igem.org/mediawiki/2014/6/6f/Oxford_Characterisation_question.png" style="float:right;position:relative; width:42%;" /> | + | <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> |
| - | <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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| - | <img src="https://static.igem.org/mediawiki/2014/7/7d/Oxford_Comparison.png" style="margin-left:0%; margin-right:0%; position:relative; width:100%;" />
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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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| | <a href="#show4" class="show" id="show4"><div class="wetlab"> | | <a href="#show4" class="show" id="show4"><div class="wetlab"> |
| - | <h1white>ATC induction of mCherry expression</h1white> | + | <h1white>How were the constructs made?</h1white> |
| | <img src="https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png" style="float:right;position:relative; width:2%;" /> | | <img src="https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png" style="float:right;position:relative; width:2%;" /> |
| | </div></a> | | </div></a> |
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| | <a href="#hide4" class="hide" id="hide4"><div class="wetlab"> | | <a href="#hide4" class="hide" id="hide4"><div class="wetlab"> |
| - | <h1white>ATC induction of mCherry expression</h1white></div></a> | + | <h1white>How were the constructs made?</h1white></div></a> |
| | <div class="list"> | | <div class="list"> |
| | <div class="white_news_block2"> | | <div class="white_news_block2"> |
| - | <h1>Wetlab data showing response in level of mCHERRY expressed with different concs of ATC</h1> | + | <img src="https://static.igem.org/mediawiki/2014/0/08/Oxford_Fran_flowchart2.png" style="float:right;position:relative; width:83%;margin-left:2%;" /> |
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| - | data analysis
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| | + | <h1>Building pOXON-1</h1><br> |
| | + | <li>The first task in the construction of the pOXON-2-dcmR-mcherry construct was the creation of pOXON-1; pME6010 with tetracycline resistance replaced by kanamycin resistance. (N.B. The KanR gene was amplified with an optimised RBS.)</li><br> |
| | + | <li>pOXON-1 was produced using the Gibson assembly method.</li><br> |
| | + | <h1>Building pOXON-2 and pOXON-2-dcmR</h1><br> |
| | + | <li>pOXON-1 was then used as the vector for the insertion of the three gblock fragment constituting the inducible expression system of dcmR via Gibson assembly.</li><br> |
| | + | <li>Upon sequencing of the product, it was determined that the version of the gblock containing the dcmR gene in the construct was actually truncated. This construct with the truncated dcmR is pOXON-2. A second Gibson assembly reaction was used to replace the truncated version with the full length gene also derived from the gblock. The resulting construct was named pOXON-2-dcmR.</li><br> |
| | + | <h1>Adding in mCherry</h1><br> |
| | + | <li>We then used pOXON-2-dcmR as the vector for the insertion of mCherry downstream of dcmR as a translational fusion by Gibson assembly.</li><br> |
| | + | <li>We therefore have a system of expressing dcmR with (pOXON-2-dcmR-mCherry) and without (pOXON-2-dcmR) the mCherry fusion in order to test whether the addition of mCherry affects the action of DcmR. Both will be submitted as BioBricks in the standard pSB1C3 backbone.</li><br> |
| | + | <li>All constructs were confirmed by sequencing.</li><br> |
| | + | <h1>Building pSRK Gm construct</h1><br> |
| | + | <li>Still under construction, we have attempted to make our second construct by inserting the pdcmAsfGFP gblock into the pSRK Gm vector by Gibson assembly. As this is proving difficult the next approach will be to insert the two components separately and to source the DNA from other sources instead of the gblock. Firstly pdcmA will be amplified from Methylobacterium extorquens DM4 genomic DNA and inserted into the pSRKGm vector. sfGFP will them be amplified from a plasmid already containing it and then added the the pSRKGm-pdcmA construct.</li><br> |
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| - | <a href="https://2014.igem.org/Team:Oxford/biosensor_characterisation"><img src="https://static.igem.org/mediawiki/2014/b/b9/Oxford_Characterisation_dark.png" style="float:left;position:relative; width:23%;" /></a> | + | <a href="https://2014.igem.org/Team:Oxford/biosensor_characterisation"><img src="https://static.igem.org/mediawiki/2014/c/ca/Oxford_Characterisation.png" style="float:left;position:relative; width:23%;" /></a> |
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| | <a href="https://2014.igem.org/Team:Oxford/biosensor_realisation"><img src="https://static.igem.org/mediawiki/2014/4/4c/Oxford_Realisation.png" style="float:right;position:relative; width:23%;" /></a> | | <a href="https://2014.igem.org/Team:Oxford/biosensor_realisation"><img src="https://static.igem.org/mediawiki/2014/4/4c/Oxford_Realisation.png" style="float:right;position:relative; width:23%;" /></a> |
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| - | <a href="https://2014.igem.org/Team:Oxford/biosensor_construction"><img src="https://static.igem.org/mediawiki/2014/a/ae/Oxford_construction.png" style="float:left;position:relative; width:23%; margin-left: 2.66%" /></a> | + | <a href="https://2014.igem.org/Team:Oxford/biosensor_construction"><img src="https://static.igem.org/mediawiki/2014/e/ef/Oxford_construction_dark.png" style="float:left;position:relative; width:23%; margin-left: 2.66%" /></a> |
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| | <a href="https://2014.igem.org/Team:Oxford/biosensor_optimisation"><img src="https://static.igem.org/mediawiki/2014/9/93/Oxford_Optimisation.png" style="float:right;position:relative; width:23%; margin-right: 2.66%" /></a> | | <a href="https://2014.igem.org/Team:Oxford/biosensor_optimisation"><img src="https://static.igem.org/mediawiki/2014/9/93/Oxford_Optimisation.png" style="float:right;position:relative; width:23%; margin-right: 2.66%" /></a> |
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| | <a href="https://2014.igem.org/Team:Oxford/biosensor"><img src="https://static.igem.org/mediawiki/2014/a/a7/Oxford_biosensor_link.png" style="float:left;position:relative;width:50%; margin-top:2%;margin-left:25%;margin-right:25%;" /></a> | | <a href="https://2014.igem.org/Team:Oxford/biosensor"><img src="https://static.igem.org/mediawiki/2014/a/a7/Oxford_biosensor_link.png" style="float:left;position:relative;width:50%; margin-top:2%;margin-left:25%;margin-right:25%;" /></a> |
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| - | <a href="https://2014.igem.org/Team:Oxford/Modelling"><img src="https://static.igem.org/mediawiki/2014/6/6e/Oxford_modelling_homepage_link.png" style="float:left;position:relative; width:50%; margin-top:2%;margin-left:25%;margin-right:25%;" /></a>
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| | Oxford iGEM 2014 | | Oxford iGEM 2014 |
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Oxford iGEM 2014
Oxford iGEM 2014
