Part:BBa_K2703000

RBCS2i1

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

1- Biological background

This is the second intron of the rubisco protein from Chlamydomonas reinhardtii.

Characterization : Sorbonne_U_Paris

BCS2i1 can be associated with a frequently used promotor, the PsaD (BBa_K1547005) (1,3) and has been studied with various others (4).

With the chassis Chlamydomonas reinhardtii, two studies (2,5) showed that transcript abundance can be enhanced up to 5,5-fold with the insertion of this intron sequence. They acquire these data with a modified antibiotic resistance gene strategy. A transgene was designed with shble, coding for the zeocin, and intron within. The more the clones were able to grow, the higher the shble gene was expressed.

To achieve such level, the position of the intron within the transgene is significant. The intron must be placed in 5’UTR for maximal enhancement, but can still be improved up to 3,7 times the targeted sequence if inserted in the CDS1. Intron sequences can be repeated or combined with other introns sequences to reach maximal enhancement. In particular, RSC2i1 can be coupled with RBCSi21. Theis and his team also showed that a smaller sequence, from 145 to 115 pb, of this part can maintain the same IME effect on transgene expression. If minimal changes on the transgene sequence length is needed, an even smaller version of this part can be use (70pb) and still improve the transgene expression by 3,8 fold.

Jaeger and his team created a software to facilitate intron-enriched DNA sequence design, compatible with MoClo’s level 0 parts : Intronserter (5). Such a tool and the IME strategy can be useful to any future IGEM team working with low expressed transgenes and any synthetic biology based project.

Intronserter website : https://bibiserv.cebitec.uni-bielefeld.de/intronserter.

Characterization was added by the 2020 Sorbonne_U_Paris team.

2020

1. Baier, T., Jacobebbinghaus, N., Einhaus, A., Lauersen, K. J. & Kruse, O. Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii. PLOS GENETICS 21.

2. Baier, T., Wichmann, J., Kruse, O. & Lauersen, K. J. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res 46, 6909–6919 (2018).

3. Schroda, M. Good News for Nuclear Transgene Expression in Chlamydomonas. Cells 8, (2019).

4. Scranton, M. A. et al. Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Research 15, 135–142 (2016).

5. Jaeger, D., Baier, T. & Lauersen, K. J. Intronserter, an advanced online tool for design of intron containing transgenes. Algal Research 42, 101588 (2019).

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).