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| | ===Applications of BBa_K2703002=== | | ===Applications of BBa_K2703002=== |
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| − | Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be use as constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution on the Chlamydomoas. It is part of our reporter system to characterize the activity and transposition rate.
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| − | <br> <br>
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| − | We couldn’t directly use our Phytobrick for the characterisation because we didn’t have time to construct all the other that are needed for the final assembly. Instead we use a slightly different Phytobrick with the same P PSAD. sequence but with one different fusion site. This part was assembled in a functional Transcription Unit with the resistance gene paromomycine that
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| − | was as a reporter gene. After transformation in C.reinhardtii D66 the functionality of the promoter was tested by counting the number of chlamydomonas colony resistant to paromomycine (15 ug/ml). Different constructions were made to characterize and compare the functionality of P pSAD . We tested different combination of promoters and 3’UTR. <br> <br>
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| − | <table>
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| − | <tr>
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| − | <td>Name</td>
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| − | <td>F1</td>
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| − | <td>Prom</td>
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| − | <td>F2</td>
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| − | <td>5'UTR</td>
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| − | <td>F3</td>
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| − | <td>Resistance</td>
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| − | <td>F4</td>
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| − | <td>3'UTR</td>
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| − | <td>F5</td>
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| − | </tr>
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| − | <tr >
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| − | <td>pCM-1</td>
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| − | <td>GGAG</td>
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| − | <td>PAR</td>
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| − | <td>TACT</td>
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| − | <td>5’UTRAR</td>
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| − | <td>AATG</td>
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| − | <td>Paromycin</td>
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| − | <td>GCTT</td>
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| − | <td>TRBCS2</td>
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| − | <td>CGCT</td>
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| − | </tr>
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| − | <tr >
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| − | <td>pCM-2</td>
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| − | <td>GGAG</td>
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| − | <td>PAR</td>
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| − | <td>TACT</td>
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| − | <td>5’UTRAR</td>
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| − | <td>AATG</td>
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| − | <td>Paromycin</td>
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| − | <td>GCTT</td>
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| − | <td>TPSAD</td>
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| − | <td>CGCT</td>
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| − | </tr>
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| − | <tr >
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| − | <td>pCM-3</td>
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| − | <td>GGAG</td>
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| − | <td>PPSAD</td>
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| − | <td>TACT</td>
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| − | <td>5’UTRPSAD</td>
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| − | <td>AATG</td>
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| − | <td>Paromycin</td>
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| − | <td>GCTT</td>
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| − | <td>TRBCS2</td>
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| − | <td>CGCT</td>
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| − | </tr>
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| − | <tr >
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| − | <td>pCM-4</td>
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| − | <td>GGAG</td>
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| − | <td>PPSAD</td>
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| − | <td>TACT</td>
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| − | <td>5’UTRPSAD</td>
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| − | <td>AATG</td>
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| − | <td>Paromycin</td>
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| − | <td>GCTT</td>
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| − | <td>TPSAD</td>
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| − | <td>CGCT</td>
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| − | </tr>
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| − | </table>
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| − | <figcaption>Figure 2: The 4 different devices made to test the functionality of the promoter PPSAD. The construction were made with the MoClo kit made for C.reinhardtii by P.Crozet et al 2018 1. </figcaption>
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| − | <img src="https://static.igem.org/mediawiki/parts/f/f1/T--Sorbonne_U_Paris--Bba_K27030032.png">
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| − | <figcaption>Figure 3: Graphic listing the 4 devices (PCM-1, pCM-2, pCM-3, pCM-4) transformed in C.reinhardtii D66 by electroporation with 100 g of DNA. The selection was made on TAP agar media with paromomycine (15 g/ml) by plate. Data are mean SD (N=3)N=3 and there were no results with a negative control.</figcaption>
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| − |
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| − | The promoter PPSAD has a slightly better activity than promoter PAR Moreover, the combination of promoter PPSAD and TPSAD seems to work better that with the terminator TRBCS2.
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| − |
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| − | </html>
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| | ===User Reviews=== | | ===User Reviews=== |
| | <!-- DON'T DELETE --><partinfo>BBa_K2703002 StartReviews</partinfo> | | <!-- DON'T DELETE --><partinfo>BBa_K2703002 StartReviews</partinfo> |
Latest revision as of 15:42, 9 October 2018
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