Difference between revisions of "Part:BBa K2703002"
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<h3> 2- Usage in iGEM projects </h3>
 
<h3> 2- Usage in iGEM projects </h3>
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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.
  
 
=='''Characterization'''==
 
=='''Characterization'''==
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table,  td {
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    border: 1px solid black;
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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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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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</html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 15:41, 9 October 2018

pSAD

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

For the silver medal criteria “Validated Part” we submit a basic part: constitutive promoter pSAD from Chlamydomonas reinhardtii. It is a high constitutive expression promoter that encodes for a ferrodoxin-binding protein of photosystem I. This pSAD promoter was submitted in 2014 by the Concordia team (Bba_K1527005). To be compatible with the PhytoBrick standard (RFC1000), an illegal BpiI restriction site was removed (A289T) 1 . We cloned P pSAD by PCR amplification in A2-A3 fusion site to use it for our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM9121 2 . We sequenced again the sequence to be sure there is no unwanted mutations.

1- Biological background

pSAD is a strong constitutive promotor in Chlamydomonas. It regulates a gene which encodes an abundant chloroplast protein located on the stromal side of the Photosystem I complex.

2- Usage in iGEM projects

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.

Characterization

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 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.

Name F1 Prom F2 5'UTR F3 Resistance F4 3'UTR F5
pCM-1 GGAG PAR TACT 5’UTRAR AATG Paromycin GCTT TRBCS2 CGCT
pCM-2 GGAG PAR TACT 5’UTRAR AATG Paromycin GCTT TPSAD CGCT
pCM-3 GGAG PPSAD TACT 5’UTRPSAD AATG Paromycin GCTT TRBCS2 CGCT
pCM-4 GGAG PPSAD TACT 5’UTRPSAD AATG Paromycin GCTT TPSAD CGCT
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.
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.
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.


References