Team:Sorbonne U Paris/Contribution
Contribution
Background:
Introns, non-coding sequences found in eukaryotic genes, can elicit an increased level of mRNA expression of any nuclear transgenes in eukaryotic organisms. This process is called Introns-mediated Enhancement (IME). Understanding this process remains a hot topic issue and the answer would have a tremendous impact in synthetic biology.
RBC2i1 (Part BBa_K2703000) is the first intron of the second small subunit of the Rubisco enzyme gene (RBSC2). It is well described and reviewed in litterature and is often used to study the IME mechanism because of the high transcription levels of the RBSC2 gene. The microalgae
Chlamydomonas reinhardtii
is a suitable model organism to study this process due to the presence of the RBCS2 gene and the high intron richness of its genome.
Bibliographic research:
RBCS2i1 can be associated with a frequently used promotor, the PsaD (BBa_K1547005) and has been studied with various others.
With the chassis
Chlamydomonas reinhardtii,
two studies showed that transcript abundance can be enhanced up to 5,5 times with the insertion of this intron sequence. They acquire these data with a modified antibiotic resistance reporter gene strategy. A transgene was designed containing the
sh
ble CDS, coding for a protein able to sequester the antibiotic zeocin. The survival rate of the colonies correlates with the transformation rate and the successful splicing (unsuccessful splicing results in cell death due to the non expression of
sh
ble). Significatively more colonies were able to grow with the rbcS2i1 intron versus the intron-less control, which indicates a IME process and a higher expression of
sh
ble.
Experimental design from Jaeger and al., Algal Research, 2019
To achieve a higher level of expression, the position of the intron within the transgene is important. The intron must be placed in the 5’UTR for maximal enhancement, but can still elicit an increase of up to 3,7 times if inserted in the CDS. Intron sequences can be repeated or combined with other introns sequences to reach maximal enhancement. In particular, RSC2i1 can be repeated up to 3 times or coupled with RBCSi2, the second intron of the RBCS gene.
Theis and his team also demonstrated that a truncated sequence of this part, from 145 to 115 pb, can maintain the same IME effect on transgene expression. If minimal impact on the transgene sequence length is needed, an even smaller version of this part can be used (70pb) and still improve the transgene expression by 3,8 times.
Jaeger and his team created a software to facilitate intron-enriched DNA sequence design, compatible with MoClo’s level 0 parts: Intronserter.
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
References:
- Merchant SS et al. . The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. (2007).
- Baier T, Jacobebbinghaus N, Einhaus A, Lauersen KJ, Kruse O. Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii. PLoS Genet. (2020).
- Baier T, Wichmann J, Kruse O, Lauersen KJ. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res. (2018).
- Schroda M. Good News for Nuclear Transgene Expression in Chlamydomonas. Cells. (2019).
- Melissa A. Scranton, Joseph T. Ostrand, D. Ryan Georgianna, Shane M. Lofgren, Daphne Li, Rosalie C. Ellis, David N. Carruthers, Andreas Dräger, David L. Masica, Stephen P. Mayfield. Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Research. (2016).
- Daniel Jaeger, Thomas Baier, Kyle J. Lauersen. Intronserter, an advanced online tool for design of intron containing transgenes. Algal Research. (2019).