Regulatory

Part:BBa_K2148016

Designed by: Chern Juin Ong   Group: iGEM16_Cambridge-JIC   (2016-10-13)
Revision as of 07:37, 19 October 2021 by MichaelCWB (Talk | contribs) (→‎References)


C.reinhardtii chloroplast rbcL 3UTR


This part contains the 3'UTR for the rbcL gene found in Chlamydomonas reinhardtii chloroplast genome. This is coded as a level-0 3UTR/TERM Phytobrick, which together with other level-0 Phytobrick can form a desired transcriptional unit for transformation.


Usage and Biology

This is a level-0 3UTR/TERM Phytobrick. It can be used along with other level-0 Phytobrick promoters and CDS to create a transcriptional unit.

Caution: In chloroplast rbcL 3'UTR don't serve as transcription terminators as in bacteria, but as elements for transcript maturation/stabilisation. atpB 3'UTR can be used as an alternative. (on advice from Prof. Saul Purton - UCL)


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 490
    Illegal XbaI site found at 478
    Illegal XbaI site found at 520
    Illegal SpeI site found at 514
    Illegal PstI site found at 28
    Illegal PstI site found at 496
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 490
    Illegal NheI site found at 42
    Illegal SpeI site found at 514
    Illegal PstI site found at 28
    Illegal PstI site found at 496
    Illegal NotI site found at 526
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 490
    Illegal BamHI site found at 508
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 490
    Illegal XbaI site found at 478
    Illegal XbaI site found at 520
    Illegal SpeI site found at 514
    Illegal PstI site found at 28
    Illegal PstI site found at 496
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 490
    Illegal XbaI site found at 478
    Illegal XbaI site found at 520
    Illegal SpeI site found at 514
    Illegal PstI site found at 28
    Illegal PstI site found at 496
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterisation

iGEM Marburg 2021 - Improvement of an Existing Part

Cell-free technology offers the possibility for high-throughput characterization of genetic parts in a shorter time frame. This timecut is notable when working with plants in general, but is even more of a hurdle when working on the chloroplast. Normally, it takes several months after a genetic device is introduced until it can be qualitatively/quantitatively characterized. The integration of a transgene in the chloroplast of an organism of choice includes very costly lab equipment and consumables. On top of this, the methods do not offer support to test multiple genetic devices in a time-efficient manner and for many plant species, no chloroplast transformation protocols are available to date.

Currently, the repertoire of available genetic parts in the field of chloroplast plant synthetic biology is hugely limited and the characterisation of these tools is very cost-intensive and time consuming. Although a handful of inducible parts and regulatory sequences exist [3] [4] [5] [6], the small collection poses a huge hurdle for more complex engineering projects. In literature, a lot of ambitious and aspirational projects have been proposed like the implementation of nitrogen fixation pathways into plants or increasing nutritional value in plants [7] [8]. But when considering the limited availability of parts, projects like these seem impossible at the current time.

The iGEM registry is one of the cornerstones of synthetic biology as it encompasses a collection of over 20.000 parts. As it is normal for iGEM projects to not be completely finished until the end of the iGEM season, we aimed to connect to the efforts of [http://2016.igem.org/Team:Cambridge-JIC iGEM Cambridge 2016]. They introduced a chloroplast part collection for the model algae Chlamydomonas reinhardtii. The toolbox included regulatory sequences, reporter genes, a CRISPR/Cas9 system and a set of homology flanks for genomic integration. As we think it is of high priority to connect to older iGEM projects and reiterate designs during future projects, we decided to have a deeper look into their parts. During a closer inspection we noticed that the 3’UTR of the rbcL gene (large subunit of the RuBisCo gene) BBa_K2148016 BioBrick of their collection was falsely linked to one of their other parts BBa_K2148007. Shortly after, we informed Vinoo Selvarajah head of the iGEM registry about this and consequently the part’s annotation has been corrected.

References

[1] Wannathong T, Waterhouse JC, Young REB, Economou CK, Purton S. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology and Biotechnology. 2016;100:5467-5477. doi:10.1007/s00253-016-7354-6.

[2] Michel Goldschmidt-Clermont, Miche`le Rahire and Jean-David Rochaix. Redundant cis-acting determinants of 3¢ processingand RNA stability in the chloroplast rbcL mRNA ofChlamydomonas. The Plant Journal (2008) 53, 566–577. doi: 10.1111/j.1365-313X.2007.03365.x

[3] Mühlbauer, S. K., & Koop, H.-U. (2005). External control of transgene expression in tobacco plastids using the bacterial lac repressor. In The Plant Journal (Vol. 43, Issue 6, pp. 941–946). Wiley. https://doi.org/10.1111/j.1365-313x.2005.02495.x

[4] Verhounig, A., Karcher, D., & Bock, R. (2010). Inducible gene expression from the plastid genome by a synthetic riboswitch. In Proceedings of the National Academy of Sciences (Vol. 107, Issue 14, pp. 6204–6209). Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.0914423107

[5] Surzycki, R., Cournac, L., Peltier, G., & Rochaix, J.-D. (2007). Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. In Proceedings of the National Academy of Sciences (Vol. 104, Issue 44, pp. 17548–17553). Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.0704205104

[6] Rojas, M., Yu, Q., Williams-Carrier, R., Maliga, P., & Barkan, A. (2019). Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes. In Nature Plants (Vol. 5, Issue 5, pp. 505–511). Springer Science and Business Media LLC. https://doi.org/10.1038/s41477-019-0412-1

[7] Li, Q., & Chen, S. (2020). Transfer of Nitrogen Fixation (nif) Genes to Non‐diazotrophic Hosts. In ChemBioChem (Vol. 21, Issue 12, pp. 1717–1722). Wiley. https://doi.org/10.1002/cbic.201900784

[8] Goicoechea, N., & Antolín, M. C. (2017). Increased nutritional value in food crops. In Microbial Biotechnology (Vol. 10, Issue 5, pp. 1004–1007). Wiley. https://doi.org/10.1111/1751-7915.12764

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