Team:Marburg/Parts

Best Basic Part

Best Basic Part

We proudly present our best new basic part: the rrn16 promoter from tobacco. We have extensively characterized this part by showing its functionality in three different species and also demonstrated that it can be easily transferred to other plant chassis. Furthermore, we have established this part as reference to allow future teams to optimize the endogenous machinery in their in-vitro systems.

Transcription In The Chloroplast

Transcription in the chloroplast is mainly driven by two different RNA Polymerases: plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP). The PEP is a bacterial-like polymerase that is a remnant of the chloroplast’s cyanobacterial ancestor and is only capable of promoting gene expression in the plastid. These polymerases are able to interact with nuclear-encoded sigma factors and therefore are able to recognize bacterial promoter motives such as the -35 (TTGACA) and the Pribnow (TATAAT) box. Similar to bacteria there are different sigma factors promoting gene expression under different growth conditions. As the PEP is structurally more sophisticated there are even more peptides involved in DNA transcription that is not fully understood yet.

The NEP is a T3/T7 phage-like polymerase that is encoded in the nucleus and is imported into the chloroplast. It was proposed that this polymerase is a remnant of a horizontal gene transfer from a bacterium to a eubacterial ancestor of today's plant cells. This type of polymerase mainly promotes gene expression in the early developmental stages of the chloroplast. In mature chloroplasts, it continues to transcribe housekeeping genes like the subunits of the plastid-encoded polymerase (rpoA, rpoB, rpoC1, and rpoC2) and proteins involved in fatty acid biosynthesis such as acetyl-CoA carboxylase (accD). In contrast, the PEP is rather active in mature chloroplasts and is primarily involved in the expression of photosynthetic genes. For other non-photosynthetic genes, motives of both polymerases can be found and it has been shown that both can promote transcription using deletion studies of important promoter sequences

The rrn16 Promoter and its Regulation

Expression of the plastid rRNA operon (rrn) is heavily regulated at the level of transcription during development. In most higher plants the rrn operon is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a σ70-type promoter with conserved −10 and −35 core promoter elements.

The 16S promoter and the PEP polymerase
The RUA motive can be seen directly upstream the -35 region of the rrn promoter promoting high levels of transcription

It has been shown that sequences upstream of nucleotide −83 do not significantly contribute to promoter function. Moreover it has been demonstrated that an essential hexameric sequence upstream of the −35 element (the rRNA operon upstream activator [RUA], GTGGGA; ) that is conserved in monocot and dicot species and that the −10 element plays only a minor role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, which is also the characteristic of strong promoters in prokaryotic chassis. It has been proposed that σ interaction with the −10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein–DNA) interaction or by protein–protein interaction between the PEP and an RUA binding transcription factor.

More Information can be accessed on our Results page

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Prrn16 Tobacco and Wheat Comparison

Moreover, we wanted to test how the 16s promoter of tobacco and wheat behave in the chloroplast cell-free extracts of both species. This experiment allows to show how compatibel the chloroplast promoters are, even if they originate from more distant plant species. Especially between monocots and dicots it has been shown, that the 16 promoter but also its regulation can differ. Furthermore, the results show that we successfully developed working endogenous transcription- translation system in wheat.

Prrn16 promoter from Tobacco and Wheat measured in cell-free systems of Tobacco and Wheat
Our teammember Michael in the lab at 5:15am performing the last cell-free measurement of this iGEM season 45minutes before the wiki freeze
The data from this measurement can be seen in the last figure (right above this picture)