Team:ETH Zurich/Experiments
From 2013.igem.org
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<br>5'-tagagacc<b>nn</b>taggatcgta<b>n</b>a<b>n</b>gtttacgcaagaaaatg | <br>5'-tagagacc<b>nn</b>taggatcgta<b>n</b>a<b>n</b>gtttacgcaagaaaatg | ||
<br>5'-tagagacct<b>n</b>taggatcgtaca<b>n</b>gtttacgcaagaaaatg | <br>5'-tagagacct<b>n</b>taggatcgtaca<b>n</b>gtttacgcaagaaaatg | ||
+ | |||
<br>Interesting versions of the promoter were sequenced and inserted into pSB1C3 backbone using custom-made oligos. They could then be used for further cloning. | <br>Interesting versions of the promoter were sequenced and inserted into pSB1C3 backbone using custom-made oligos. They could then be used for further cloning. | ||
</td> | </td> | ||
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<tr> | <tr> | ||
- | <td> | + | <td>4</td> |
<td>Receiver cell construct for GFP experiments with positive feedback loop to reduce leakiness</td> | <td>Receiver cell construct for GFP experiments with positive feedback loop to reduce leakiness</td> | ||
- | <td>BBa_J09855-BBa_E0840 construct with the pLac promoter replaced with pLuxR to build a positive feedback loop. The promoter was inserted with two pairs of custom-made oligos using XbaI and HindIII restriction | + | <td>BBa_J09855-BBa_E0840 construct with the pLac promoter replaced with pLuxR to build a positive feedback loop. The promoter was inserted with two pairs of custom-made oligos using XbaI and HindIII restriction sites. |
- | < | + | <br>Oligos: |
+ | <br>5’- Ctagagacctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaatactaga | ||
+ | <br>5’- gattaaagaggagaaatactagatgaaaaacataaatgccgacgacacatacagaataattaataaaattaa | ||
+ | <br>5’- AATCTCTAGTATTTATTCGACTATAACAAACCATTTTCTTGCGTAAACCTGTACGATCCTACAGGTCT | ||
+ | <br>5’- AGCTTTAATTTTATTAATTATTCTGTATGTGTCGTCGGCATTTATGTTTTTCATCTAGTATTTCTCCTCTTT | ||
+ | </td> | ||
+ | <td>[[File:Pla6.png|300px]]</td> | ||
</tr> | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>Receiver cell construct with mutated pLux promoter library and RFP reporter to test together with wild-type pLux GFP receiver cells in one cell</td> | ||
+ | <td>Library of mutated pLux promoters (see above) backbone (SpeI,PstI) and RFP (SpeI,PstI) insert from [http://parts.igem.org/Part:BBa_J23118 BBa_J23118]</td> | ||
+ | <td>[[File:Pla7.png|225px]]</td> | ||
+ | </tr> | ||
+ | |||
</table> | </table> |
Revision as of 10:36, 4 October 2013
Contents |
Final Circuit
For the final Colisweeper circuit we plan a four plasmid system. The mine cells constitutively express LuxI for signal generation and NagZ as identifier hydrolase. In the non-mine cells LuxR is expressed constitutively to process the OHHL signal. PhoA is expressed constitutively as well as reporter for safe cells. Aes and GusA are expressed from pLux promoters with different sensitivities. You can find all the biobricks we used and our own new biobricks in the figure below.
Figure 1. Plasmids in mine and non-mine cells: move the cursor over the separate parts to check which biobricks we used.
Cloned Constructs
To get to the circuit mentioned above we tested different versions of the circuit. For example we started our experiments using GFP as a reporter instead of the hydrolases. Then we also tested different LuxI and LuxR generating constructs. In the following table we list all the biobricks we used, the plasmids we cloned and what experiments we used them for. In general we used standard biobrick cloning techniques as described in the methods section. Whenever we used PCR gene amplification for cloning, we list the primers used in the following table. To be able to co-transform different plasmids we used backbones with compatible origins of replication and resistance genes. In the table you can find which backbone versions we used for which constructs.
pLux constructs | |||
---|---|---|---|
Description | Cloning | Maps | |
Hydrolase constructs | |||
---|---|---|---|
Description | Cloning | Maps | |
Aes coding region with RBS in SB1C3 backbone | BBa_K1216002 |
Groupparts / Biobricks
In the following table you can find all all the biobricks that were submitted by our group.
<groupparts>iGEM013 ETH_Zurich</groupparts>