Part:BBa_K2703002

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_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.

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.

Name	F1 : A2	PART	F2: A3
pSAD_A2-B3	TGAC	PPSAD	TACT

Figure 1 : Schematic representation of the Phytobrick BBa_K2703002 and its fusion site for the MoClo assembly. It complies with the Common Genetic Syntax fusion sites.

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

1. 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

DOI: 10.1021/acssynbio.8b00251


2. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, (2011).