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| | <h1>Kill-Switch</h1> | | <h1>Kill-Switch</h1> |
| - | s that we made here .
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| - | During the project there was a close collaboration with the modelling part of our project. The system was modelled and its conc
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| - | <p>
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| - | When the organism cannot sense rhamnose anymore the third state of the kill-switch is induced. In this final state the pCIλ/Tet promoter is not repressed by the CIλ repressor or the TetR repressor leading to the expression GFP. In the final system this GFP will be a toxin that will kill the cell.
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| - | Besides the self-destruction system the kill-switch circuit has lots of potential applications. The regulatory properties of this system can be utilized for any genetic system that needs to memorize the input signal and express the output gene when this signal is gone. The input promoter can be changed into the sensing promoter of choice. Likewise, the output gene can be changed giving the system a great flexibility. An even stronger improvement would be the addition intercellular communication, making a collective synchronized memory possible, which enables simple control over vast populations of bacteria equipped with this system.
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| - | </p>
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| - | <h2 id="ioplasmid">The Input Output Plasmid</h2>
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| - | <p>
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| - | The input/output plasmid is the plasmid that can sense rhamnose and produce GFP or toxin, see Figure 5.
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| - | </p>
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| - | <figure>
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| - | <img src="https://static.igem.org/mediawiki/2014/2/2f/Wageningen_UR_killswitch_Pic5.png" width="80%">
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| - | <figcaption> Figure 5: the overview of the kill switch regulatory system. The input output plasmid is circled.
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| - | </figcaption>
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| - | </figure><br/>
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| - | <p>
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| - | Figure 10 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states. Thus, the toggle switch is able to choose a random state and no state is significantly more preferred than the other . This is in contrast to what the model predicted.
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| - | </p>
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| - | <p>
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| - | <b>Rhamnose mediated characterization parts</b>
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| - | </p>
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| - | <figure>
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| - | <figcaption>
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| - | Table 3: All parts that were made for the rhamnose mediated characterization and send to the registry by the kill-switchproject. The first column contains the biobrick code the second column explains the function of the biobrick and the last Coolum explains where it is used for in the kill-switchproject.
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| - | </figcaption>
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| - | </figure>
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| - | <p>
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| - | <b>Parts we used</b>
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| - | </p>
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| - | <figure>
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| - | <figcaption>
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| - | Table 4: All parts that we used form the registry in the kill-switchproject. The first column contains the biobrick code the second column explains the function of the biobrick and the last Coolum explains where it is used for in the kill-switchproject.
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| - | </figcaption>
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| - | </figure>
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| - | <br/>
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| - | <h2 id="references">References</h2>
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| - | <ol>
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| - | <li>Gardner, T.S., C.R. Cantor, and J.J. Collins, Construction of a genetic toggle switch in Escherichia coli. Nature, 2000. 403(6767): p. 339-42.</li>
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| - | <li>Cox, R.S., 3rd, M.G. Surette, and M.B. Elowitz, Programming gene expression with combinatorial promoters. Mol Syst Biol, 2007. 3: p. 145.</li>
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| - | <li>Lanzer, M. and H. Bujard, Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A, 1988. 85(23): p. 8973-7.</li>
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| - | <li>Kelly, J.R., et al., Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng, 2009. 3: p. 4.</li>
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| - | </ol>
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| | </section> | | </section> |
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