Part:BBa_K4608005
Truncated Csx30 (trCsx30)
Codes for a truncated version of Csx30, consisting of only amino acids 396 - 565. Used as the versatile cleavage domain in all of our fusion proteins. Codon optimised for expression in yeast.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
This BioBrick Part corresponds to the truncated Csx30 protein that the 2023 McGill iGEM team used for their research project, Proteus. For more details on the background of our project and prior information, please visit: https://2023.igem.wiki/mcgill/.
Note that this part is about truncated Csx30, from amino acids 396 to 565, without the methionine start codon. In Proteus, we used truncated Csx30 and inserted it in the linker of human gasdermin D and E. We used full length Csx30 as a control, to test for yeast cytotoxicity and cleavage by native caspases. We also included full-length Csx30 as a separate part, for teams interested in creating their own fusion proteins in future projects.
Background
This 64 kDa protein that exists in bacteria, and is part of the Cas7-11 and Csx29 system as defense against bacteriophage DNA. Csx30 has a domain from residues 396-565 where the TPR-CHAT protease Csx29 binds and subsequently cleaves at residues 427-429. It is possible to truncate Csx30 to just the 19 kDa binding domain from residues 396-565 allowing for easier insertion into a fusion protein. In Proteus, this was the natural linker between gasdermin N- and C-termini. The specific domain in Csx30 is required for cleavage by Csx29, thus preventing any off-target protein cleavage by Csx29. In brief, Csx30 is a protein with a specific domain that is recognized by Csx29, after which it is cleaved nearby the recognition domain. These domains together are in truncated Csx30. This protein is a core component of the Craspase system, and links transcriptome detection and sensing with an effector change at the protein level. It is fusion proteins using truncated Csx30 that is the real syn-bio aspect of the Proteus, and the crux of our project.
Figure 1 | PDB structure (8EEY) crystal structure of Csx30 protein bound to Csx29 and Cas7-11 (Both not shown). The protein fold shown was the only part of Csx30 that was able to crystallize consistently, and consists of amino acids 407 to 560.
Parts assembly
Cloning truncated Csx30 into GSDM in pYes:
BioBrick characterization=
Truncated Csx30 was never expressed on its own, without being part of a larger Csx30-Gasdermin fusion proteion. Validation of truncated Csx30 will therefore be the same as that of the fusion proteins, since it’s the core, functional componant.
Once we constructed all of our fusion proteins, we built up a yeast strain that expressed all the Craspase components (a KRAS G12D gene, dCas7-11, Csx29, and a KRAS G12D-targeting gRNA) and transformed each fusion protein individually into this strain. These final strains would have all the components to test whether the fusion proteins could get cleaved by the Craspase system. By the iGEM wiki freeze, we were only able to successfully transform three of the fusion proteins, GDDx30 N-term, GDDx30 C-term, GDDx30 30% replaced, and so these are the fusion proteins we ended up testing. Tests of the remaining fusion proteins are ongoing.
To determine whether or not the fusion proteins in the strains we had built up were capable of causing cell death, we decided to do a live death assay by staining our strains with fluorescein diacetate (stains alive cells green) and propidium iodide (stains dead cells red), then subsequently imaging these cells using confocal microscopy. This would be a rapid way we could induce our cultures to express all the parts of our system, and then induce the transcription of KRAS (with cyanamide) to see if our system was able to cause cell death. With the help of Dr. Ayyappa Perumal at McGill University, we were able to rapidly image the samples using a confocal microscope and generate qualitative and quantitative information about the validity of our system found below.
Qualitatively, we saw that the GDDx30 C-term fusion protein caused significant cell death (represented by red cells) compared to the control sample that did not express any fusion protein. Qualitatively, we saw that the GDDx30 C-term fusion protein caused significant cell death (represented by red cells) compared to the control sample that did not express any fusion protein.
 Control yeast sample, containing the full system but lacking expression of any fusion protein. Little cell death is observed, indicating that the Craspase components themselves are not significantly toxic. |
 Yeast containing the full system and expressing GDDx30 C-term. Significant yeast cell death is observed, indicating that this fusion protein may have worked. |
Quantitatively, when we used ImageJ to quantify the number of red cells over the total number of cells in our microscopy photos, we see a significant decrease in cell viability for both our positive control (GSDME truncated to just its N-terminus, which is toxic to the cell) as well as the GDDx30 C-term fusion protein.
 Replicate 1 of microscopy results, using a fully complimentary gRNA to the target RNA that should activate the Craspase system. |
 Replicate 1 of microscopy results, using a gRNA that contains one mismatch to the target RNA that should theoretically not activate the Craspase system. |
 Replicate 2 of microscopy results, using a fully complimentary gRNA to the target RNA that should activate the Craspase system. |
 Replicate 2 of microscopy results, using a gRNA that contains one mismatch to the target RNA that should theoretically not activate the Craspase system. |
Furthermore, we wanted to confirm that our toxic fusion proteins were being cleaved, and that this was the reason behind toxicity. We grew liquid cultures of our full system yeast expressing our FLAG-tagged fusion protein, induced expression of all Proteus components using Gal and cyanamide-induction, and subsequently ran lysates of these induced cultures on an SDS page, for western blot. We then stained the membrane containing these lysates using a FLAG tag antibody, to probe for the size of our fusion proteins. If the fusion proteins were expressed and cleaved, we would expect to see fragment sizes at the following sizes:
| Fusion protein | Full length (uncleaved) | Tagged fusion protein (cleaved) fragment length |
| GDDx30 N-term | 73 kDa | 32 kDa |
| GDDx30 C-term | 73 kDa | 36 kDa |
| GDDx30 30% replaced | 72 kDa | 33 kDa |
Upon analysis of the blots, we were able to confirm successful expression of all of our three fusion proteins that we tested, which showed up at their expected sizes of ~75 kDa each. The band corresponding to the full length version of each fusion protein is in blue. Two duplicates of our negative control, with no FLAG-tagged proteins being expressed (lanes 5 and 6 from the left), showed negligible background staining, validating the specificity of our FLAG tag antibody.
We observed that for both replicates of GDDx30 30% replaced and GDDx30 N-term, there was a band at the expected size (33 and 32 kDa, respectively) for cleavage. These bands are circled in red, and these results suggested to us that the GDDx30 N-term and 30% replaced fusion proteins were indeed being cleaved by the Craspase system inside yeast! This means truncated Csx30 was successfully cleaved by Csx29 in this fusion protein.
Intriguingly however, we saw no band at around 36 kDa indicating cleavage of GDDx30 C-term. Furthermore, we also did not see a band at the expected size of our positive control, GSDME N-term. This second observation was quite confounding at first, because the viability microscopy assays clearly showed significant cell death for our positive control. Furthermore, this problem extended to GDDx30 C-term, which we observed by microscopy to also be toxic to the yeast.
Upon reviewing the literature of Gasdermins however, we found an explanation. In multiple papers (Ji et al., 2023; Rogers et al., 2017; Lei et al., 2027; Liu et al., 2016), whenever the toxic N-terminus of a gasdermin is expressed in cells (in both yeast and mammalian), there seems to be little to no protein detectable in the cell lysate, which consists mostly of intracellular proteins. Instead, the gasdermin N-terminus can be found in the medium the cells were grown in, suggesting that the toxic gasdermins are secreted/”shed” once they oligomerize and form membrane pores. Therefore, these western blots further supported our proof of concept results, suggesting that truncated Csx30 is successfully being cleaved, thus toxic N-terminal fragments of GDDx30 C-term and GSDME N-terminus embedded themselves in the membrane, forming oligomeric pores that could not be detected in the cell lysate. This would explain the lack of bands at the expected size for N-terminally truncated GSDME and cleaved GDDx30 C-term. We encourage other teams and researchers to build on these findings to test the Proteus system in other host systems, such as mammalian, and continue to explore the limitless possibilities with Craspase.
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