Plant Thiamine Pyrophosphate Riboswitch
The idea behind this part
Our goal was the design of a plant system that would be able to detect microcystin-LR (a cyanotoxin abundant in eutrophic waters) and enable the expression of a phosphate transporter as a response (thus alleviating the phenomenon). The mechanism underlying this system was a synthetic riboswitch. To build this new riboswitch, we tried to imitate the function of the only naturaly found riboswitch known to biologists: the thiamine pyrophosphate (TPP) riboswitch. By combining the sequence of a microcystin-LR targeting aptamer and the TPP riboswitch in a rational manner, we sought to develop new ones. Future iGEM teams might find this process (1) useful for their projects, should they wish to engineer a plant to detect similar molecules.
This part may also be used as a simple TPP inducable NOT gate in the place of a terminator (or 3' UTR - depending on the assembly method syntax) without making any changes to the aptamer region (2). However, there is an important caveat (explained below).
For the scope of our project, we would use the TPP riboswitch to establish a proof of concept before testing the new ones. Things did not go according to plan though, and we did not have a proper chassis to test the part. This is because, for the experiments to take place, we should be in full control of the TPP content in the plant. Otherwise, the plant's own TPP concent will interfere with the one added. This means that the plant must be TPP deficient (either by being a mutant or with RNAi targeting a TPP biosynthesis enzyme), and all TPP must be administered exogenously. Thus, it is highly recommended that if you want to use (and/or characterize) this part, you must be quick to find seeds for a TPP auxotroph plant strain!
Ancient relics in modern chassis
BiologyOnce the sequence of the microcystin-LR aptamer was found (3), we had to think of a way to incorporate it into a genetic control device. Aptamers, as far as we know, do not exist in organisms as free molecules. They are, instead, parts of riboswitches: one of the oldest mechanisms devised by primordial life forms to regulate metabolic pathways.
What exactly are riboswitches, then? A riboswitch is a regulatory segment of an RNA that binds either a metabolite, a small molecule or an ion, resulting in a change in the production of the protein that it encodes. The regulatory effect can be exerted on transcription, splicing, mRNA stability, and translation. The ligand binds to the aptamer region of the riboswitch. Often, another domain near the aptamer, called expression platform, is able to undergo conformational changes that enable the riboswitch to interact with DNA elements that control genes involved in the production of the ligand that bound the riboswitch. They can have either repressive or stimulating action. While very widespread among prokaryotes, there seems to be a very limited number of candidates in eukaryotes.
So, they have been found in eukaryotes. A particular one, the thiamine pyrophosphate (hereafter TPP) riboswitch, was found in bacteria, fungi, and many phototrophic organisms, from (evolutionary) Lower microalgae to, almost all, Higher plants (4). They are probably the same one, that has evolved from an early age into all these eukaryotic kingdoms, with minor differences (5). It is a riboswitch that regulates the biosynthesis of TPP. In Higher plants, it works by affecting mRNA splicing. It is located inside an intron, in the 3΄ UTR of key enzymes in the TPP biosynthetic pathway. As soon as TPP binds to the aptamer region, the rest of the sequence changes conformation to allow the splicosome to bind and splice the intron away, removing the poly-A signal. This causes mRNA instability and degradation.
1. This is a plant-originating part. 2. It can act as a TPP-inducible NOT gate. 3. It can be engineered to detect different ligands (at least in silico; in vivo and in vitro functionality remains to be tested). 4. It is a splicing riboswitch, acting on the pre-mRNA level. 5. Get a TPP auxotroph plant strain before use (and of course, buy TPP to add while conducting experiments). This note is irrelevant if you try to incorporate the part in other organism types (e.g. mammalian cells).
Sequence and Features
Here is the whole 3'UTR sequence of the A. thalaliana THIC gene mRNA. The 5' and 3' splicing sites of the intron of interest, as well as the poly-A signal that gets spliced out in the presence of ligands, is depicted. The riboswitch region is also visible.
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal XhoI site found at 578
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
1. https://2022.igem.wiki/thessaly/engineering 2. Bocobza, S. et al., (2007). Riboswitch-dependent gene regulation and its evolution in the plant kingdom. Genes & development, 21(22), 2874–2879. 3. Ng, A., et al., (2012). Selection, characterization, and biosensing application of high affinity congener-specific microcystin-targeting aptamers. Environmental science & technology, 46(19), 10697–10703 4. Bocobza, S. E., & Aharoni, A. (2014). Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae. The Plant journal : for cell and molecular biology, 79(4) 5. Barrick, J. E., & Breaker, R. R. (2007). The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome biology, 8(11), R239.