Difference between revisions of "Part:BBa K3758200"

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psbA 3-UTR (Nicotiana tabacum)

Part Description

This part was created using the Phytobrick Entry Vector with GFP dropout BBa_K2560002 and was designed to be compatible with the Phytobrick assembly standard. All parts created this year were acquired via PCR from purified DNA samples using a CTAB based method ^[1], by primer annealing, primer annealing, and extension reactions or synthesized via IDT/Twist.

All parts this year were produced to be used in the chloroplast of different plant species. For the characterization of these parts they were tested in chloroplast cell-free systems (ccfs) from either the same species or they were tested in ccfs from other plant species. Plastid parts offer the benefit of highly conserved regulatory sequences that can be used across species. Although characterizing chloroplast parts is a huge effort, in literature, it has been shown that plastid parts can be used across species to drive gene expression ^[2]. We believe that based on this knowledge we can create valuable parts that can be screened for activity in our system with the final goal of building a variety of different parts. This collection shall help combat unwanted recombination events in vivo that sometimes impede the successful functionality of the genetic design.

3’UTR

3’ untranslated regions do not function as efficient transcription terminators. They rather form stem-loop RNA secondary structures that are thought to protect the mature RNA from 3’-5’ exoribonucleases. While 3’UTRs play a strong role in the accumulation of RNA transcripts ^[11], their effect on protein accumulation is in most cases rather limited ^[12]. Nonetheless, under specific conditions and depending on the protein that is planned to be expressed, the choice of the 3’UTR may play a bigger role in protein expression. Similarly to the 5’UTR, some 3’UTRs are trimmed by specific pentatricopeptide repeat (PPR) proteins that are imported from the nucleus of the plant. ^[13].

Characterization & Measurement

Results

Marburg collection 3.0

Sequence and Features

References

[1] Aboul-Maaty, N. A.-F., & Oraby, H. A.-S. (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43(1). https://doi.org/10.1186/s42269-019-0066-1

[2] Fuentes, P., Zhou, F., Erban, A., Karcher, D., Kopka, J., & Bock, R. (2016). A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. ELife, 5. https://doi.org/10.7554/elife.13664

[3] Filée, J., & Forterre, P. (2005). Viral proteins functioning in organelles: a cryptic origin? Trends in Microbiology, 13(11), 510–513. https://doi.org/10.1016/j.tim.2005.08.012

[4] Liere, K., & Börner, T. (2007). Transcription and transcriptional regulation in plastids. In Cell and Molecular Biology of Plastids (pp. 121–174). Springer Berlin Heidelberg. https://doi.org/10.1007/4735_2007_0232

[5] Xie, G., & Allison, L. (2002). Sequences upstream of the YRTA core region are essential for transcription of the tobacco atpB NEP promoter in chloroplasts in vivo. Current Genetics, 41(3), 176–182. https://doi.org/10.1007/s00294-002-0293-z

[6] Kuroda, H., & Maliga, P. (2002). Overexpression of the clpP 5′-Untranslated Region in a Chimeric Context Causes a Mutant Phenotype, Suggesting Competition for a clpP-Specific RNA Maturation Factor in Tobacco Chloroplasts. Plant Physiology, 129(4), 1600–1606. https://doi.org/10.1104/pp.004986

[7] Klinkert, B. (2006). Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon. Nucleic Acids Research, 34(1), 386–394. https://doi.org/10.1093/nar/gkj433

[8] Zhelyazkova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., & Barkan, A. (2011). Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. Nucleic Acids Research, 40(7), 3092–3105. https://doi.org/10.1093/nar/gkr1137

[9] Zoschke, R., Kroeger, T., Belcher, S., Schöttler, M. A., Barkan, A., & Schmitz-Linneweber, C. (2012). The pentatricopeptide repeat-SMR protein ATP4 promotes translation of the chloroplastatpB/EmRNA. The Plant Journal, 72(4), 547–558. https://doi.org/10.1111/j.1365-313x.2012.05081.x

[10] Zhang, L., Zhou, W., Che, L., Rochaix, J.-D., Lu, C., Li, W., & Peng, L. (2019). PPR Protein BFA2 Is Essential for the Accumulation of the atpH/F Transcript in Chloroplasts. Frontiers in Plant Science, 10. https://doi.org/10.3389/fpls.2019.00446

[11] Tangphatsornruang, S., Birch-Machin, I., Newell, C. A., & Gray, J. C. (2010). The effect of different 3′ untranslated regions on the accumulation and stability of transcripts of a gfp transgene in chloroplasts of transplastomic tobacco. Plant Molecular Biology, 76(3–5), 385–396. https://doi.org/10.1007/s11103-010-9689-1

[12] Eberhard, S., Drapier, D., & Wollman, F.-A. (2002). Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. The Plant Journal, 31(2), 149–160. https://doi.org/10.1046/j.1365-313x.2002.01340.x

[13] Pfalz, J., Bayraktar, O. A., Prikryl, J., & Barkan, A. (2009). Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts. The EMBO Journal, 28(14), 2042–2052. https://doi.org/10.1038/emboj.2009.121

[14] Suzuki, J. Y., Sriraman, P., Svab, Z., & Maliga, P. (2003). Unique Architecture of the Plastid Ribosomal RNA Operon Promoter Recognized by the Multisubunit RNA Polymerase in Tobacco and Other Higher Plants. The Plant Cell, 15(1), 195–205. https://doi.org/10.1105/tpc.007914

[15] Occhialini, A., Piatek, A. A., Pfotenhauer, A. C., Frazier, T. P., Stewart, C. N., Jr., & Lenaghan, S. C. (2019). MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology. Plant Physiology, 179(3), 943–957. https://doi.org/10.1104/pp.18.01220