Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
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_K1547005). 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.
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.
|Name||F1 : A2||PART||F2: A3|
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.
- Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii Pierre Crozet, Francisco J. Navarro, Felix Willmund, Payam Mehrshahi, Kamil Bakowski, Kyle J. Lauersen, Maria-Esther Pérez-Pérez, Pascaline Auroy, Aleix Gorchs Rovira, Susana Sauret-Gueto, Justus Niemeyer, Benjamin Spaniol, Jasmine Theis, Raphael Trösch, Lisa-Desiree Westrich, Konstantinos Vavitsas, Thomas Baier, Wolfgang Hübner, Felix de Carpentier, Mathieu Cassarini, Antoine Danon, Julien Henri, Christophe H. Marchand, Marcello de Mia, Kevin Sarkissian, David C. Baulcombe, Gilles Peltier, José-Luis Crespo, Olaf Kruse, Poul-Erik Jensen, Michael Schroda, Alison G. Smith, and Stéphane D. Lemaire ACS Synthetic Biology 2018 7 (9), 2074-2086
- Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, (2011).