Part:BBa_K5090000

Codon-Optimized GST-Argonaute 2

Description

Argonaute2 (Ago2) is a protein that functions as the catalytic center of the RNA-Induced Silencing Complex (RISC) in humans (Shiekhattar et al., 2005). As discussed by Shiekhattar et al., it achieves its regulation in a few steps. First, Ago2 binds to a double-stranded microRNA, separating the microRNA into a guide strand and passenger strand. Then, Ago2 allows its guide microRNA strand to locate it toward a complementary target site on strands of mRNA. Finally, Ago2 binds the microRNA and mRNA strands, and performs a cleavage such that the mRNA is no longer able to be bound by a ribosome to synthesize proteins (Shiekhattar et al., 2005). Such that this part would be functional in non-Rosetta E. coli, we added a GST tag (BBa_K2719000) in the 5' direction of the Ago2 sequence for stabilization. The whole protein should be 123kDa , which is the combined molecular weight of GST (GST-tagged Proteins–Production and Purification - US, n.d.) which is 26 kDA and Ago2 (“Argonaute2”, 2018) which is 97 kDa.

This specific part was created using IDT’s codon optimization tool. We used the amino acid sequence for Human Ago2 Isoform 1 found here (https://www.ncbi.nlm.nih.gov/protein/NP_036286.2?report=fasta), and applied the following settings: -Sequence Type: DNA -Product Type: gBlocks Gene Fragments -Organism: Escherichia coli

Usage and Biology

Argonaute2 can be utilized to repress certain genes, thus manipulating their expression. One may elect to add regulatory sequences in the 5’ UTR of Argonaute2 to allow its repression to be selective. In the Micronaut gene circuit of the 2024 Stony Brook University iGEM Team, we utilized Argonaute 2 as a component that would inhibit the expression of the regulatory proteins LacI and L7Ae, which would otherwise be themselves inhibiting GFP expression.

We built upon the advancements of Laird et al. (2017) and Wang et al. (2023) by implementing their enhanced LacI (our LacI part link here), which was developed by Laird et al., and L7Ae (our L7Ae part link here), as a dual regulation system in a bacterial context. This is different from the mammalian implementation. Specifically, we implemented the two repressors in E. coli-based S30 cell-free system as part of a gene circuit that includes a GFP which is regulated by two lac operators (BBa_K079017) for LacI and a kink turn (BBa_K4140015) for L7Ae. In our circuit, as in the mammalian circuit created by Wang et al., LacI and L7Ae are linked by P2A (see note below) (link to our P2A w/ GSG linker; from DR paper), which as the “self-cleaving peptide” can cleave proteins being synthesized by the ribosome as they are being produced into distinct units. With this, we are able to express LacI and L7Ae as close to the same frequency as possible.

Additionally, the combined LacI-P2A-L7Ae coding sequence is preceded, in three different configurations, by a target complementary site for microRNA-326. With this target site and in the presence of microRNA-326, Argonaute2, which is expressed as a fusion protein GST-Ago2 (put combined GST-Ago part here) can bind to microRNA-326 and be guided to the target site exposed on an mRNA strand, at which point it will make a cleavage and cut the mRNA before it can lead to protein expression. Thus, LacI and L7Ae, which regulate GFP expression, are themselves regulated by Argonaute2 and microRNA-326. The practical use of this is so that our system is able to detect microRNA-326, with fluorescence only occurring in the presence of that specific oligonucleotide. It is possible to swap out microRNA-326 with another microRNA so that this system could be utilized to detect different microRNAs.

Characterization

Wang et al. also characterized the effectiveness of this L7Ae in their mammalian systems. Specifically, they set up an L7Ae sequence preceded by a specific microRNA sequence to regulate its expression. With the transfection of different microRNAs, the cells transfected with the incorrect exhibited relative firefly luciferase expression nearly 1 fourth the amount of the cells which were transfected with the correct microRNA and thus had their L7Ae repressed. This not only confirmed that their L7Ae was achieving repression, but also that it could be deactivated with microRNA through the cleaving mechanism of Ago2, which is ordinarily present in mammalian cells.

In our system, characterization was performed in combination with Argonaute2-based regulation of this construct and measured through relative GFP expression. We gathered multiple measures of the effectiveness of LacI and L7Ae.

Wet Lab

In Wet Lab, we were able to confirm that Ago2 was successfully cloned into E. coli. Our sequencing results demonstrate that this cloning occurred with two mutations, one silent mutation that changed a codon retained coding for Glycine, and one missense that switched a Leucine to Isoleucine. We decided not to pursue site-directed mutagenesis for this missense mutation as Leucine and Isoleucine are both amino acids with hydrophobic side chains. Additionally, analysis on Uniprot identified that the leucine/isoleucine is not present in a major conserved specific region of Argonaute2, though it is present in a major conserved domain (“Uniprot”, n.d.).

Figure 1: Plasmid derived from pJL1 into which codon-optimized Ago2 was successfully cloned. In E. coli, this would not on its own be a functional part.

We further cloned the GST tag upstream of Ago2 to complete the creation of a single functional unit for use in E. coli-based systems. This cloning was successful and had no additional mutations.

Figure 2: Plasmid derived from pJL1 into which GST was cloned, making the complete basic functional part Codon-Optimized GST-Ago2.

To verify expression, we conducted a Coomassie stain and Western blot. Unfortunately neither yielded results. As you may see in the picture below for our Coomassie, no GST-Ago2 appeared in the range between 100kDa and 150kDa (the second and third bars from the top, respectively), as would have been expected. Similarly for the picture of our Western Blot below, a band would have appeared in the lane between the two ladders but it did not. As these attempts were conducted within the context of in vivo expression in E. coli, we are also attempting to express in S30 cell-free systems and verify with Coomassie and Western. These tests are ongoing at the time of writing.

Figure 3: Coomassie from August 2024, in which GST-Ago2 did not appear.

Figure 4: Western from August 2024, in which GST-Ago2 did not appear.

Additionally, we are going to verify whether or not the addition of Ago2 to an S30 in which genes are regulated under complementary miRNA target sites is successful in suppressing those genes. This is also ongoing at time of writing.

Dry Lab

In Dry Lab, we were able to characterize that in the presence of microRNA-326, Ago2 should be able to reactivate GFP expression through its suppression of LacI and L7Ae, which are under regulation of a microRNA-326 target site. This should generally hold true for other linked microRNAs and target sites, so long as the sequences are complementary.

Specifically, given an in-vivo context in E. coli, expression should range from the concentration of approximately 2.5*10^-4 M without any dual regulation, nearly 7*10^-10 M with dual-regulation, and 2.5*10^-4 once again when Ago2 and microRNA-326 are present to facilitate repression of the dual-regulation system.

Figures 5, 6, and 7: Graphs illustrating the effect of Ago2 upon GFP expression given GFP is placed under LacI and L7Ae regulation and LacI and L7Ae are placed under regulation of Ago2 and miRNA-326.

Note: In the paper by Wang et al., the authors misidentify P2A as T2A. NCBI BLAST analysis of the sequence they provide for what they call T2A reveals that it is P2A.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3133
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1984
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1056
  Illegal SapI.rc site found at 85
  

References

• Argonaute 2 (a.a. 389-398) Antibody. (2018). ECM Biosciences. https://ecmbio.com/products/ap5281

• GST-tagged Proteins–Production and Purification - US. (n.d.). Www.thermofisher.com. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gst-tagged-proteins-production-purification.html

• Gregory, R. I., Chendrimada, T. P., Cooch, N., & Shiekhattar, R. (2005). Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing. Cell, 123(4), 631–640. https://doi.org/10.1016/j.cell.2005.10.022

• Lee, K.-H., Oghamian, S., Park, J.-A., Kang, L., & Laird, P. W. (2017). The REMOTE-control system: a system for reversible and tunable control of endogenous gene expression in mice. Nucleic Acids Research, 45(21), 12256–12269. https://doi.org/10.1093/nar/gkx829

• Rivas, F. V., Tolia, N. H., Song, J.-J., Aragon, J. P., Liu, J., Hannon, G. J., & Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Structural & Molecular Biology, 12(4), 340–349. https://doi.org/10.1038/nsmb918

• Salvatore, V., Potenza, N., Papa, U., Nobile, V., & Russo, A. (2010). Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies. International Journal of Molecular Sciences, 11(2), 745–753. https://doi.org/10.3390/ijms11020745

• Shu, W.-J., Lee, K., Ma, Z., Tian, X., Jong Seung Kim, & Wang, F. (2023). A dual-regulation inducible switch system for microRNA detection and cell type-specific gene activation. Theranostics, 13(8), 2552–2561. https://doi.org/10.7150/thno.84111

• UniProt. (2024). Uniprot.org. https://www.uniprot.org/uniprotkb/Q9UKV8/entry#family_and_domains