Difference between revisions of "Part:BBa K2148016"

Revision as of 07:31, 19 October 2021

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 [1] [2] [3] [4], 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 [5] <nowiki [6</nowiki>]. But when considering the limited availability of parts, projects like these seem impossible at the current time.

References

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.

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

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-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 [https://doi.org/10.1111/j.1365-313X.2005.02495.x<nowiki>[1]</nowiki>] [https://doi.org/10.1073/pnas.0914423107<nowiki>[2]</nowiki>] [https://doi.org/10.1073/pnas.0704205104<nowiki>[3]</nowiki>] [https://doi.org/10.1038/s41477-019-0412-1<nowiki>[4]</nowiki>], 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 [<href="https://doi.org/10.1002/cbic.201900784">5</a>, <a href="https://dx.doi.org/10.1111%2F1751-7915.12764">6</a>]. But when considering the limited availability of parts, projects like these seem impossible at the current time.
+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 [https://doi.org/10.1111/j.1365-313X.2005.02495.x<nowiki>[1]</nowiki>] [https://doi.org/10.1073/pnas.0914423107<nowiki>[2]</nowiki>] [https://doi.org/10.1073/pnas.0704205104<nowiki>[3]</nowiki>] [https://doi.org/10.1038/s41477-019-0412-1<nowiki>[4]</nowiki>], 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 [https://doi.org/10.1002/cbic.201900784<nowiki>[5]</nowiki>] [https://dx.doi.org/10.1111%2F1751-7915.12764<nowiki[6]</nowiki>]. But when considering the limited availability of parts, projects like these seem impossible at the current time.
 </p>