Part:BBa_K5090002

L7Ae

Description

L7Ae is a translational repressor that originated in Archaea (Saito et al., 2009). It achieves this translational repression by binding to the kink turn operator (BBa_K4140015) in an mRNA sequence, preventing the ribosome from performing translation of that mRNA sequence into a protein. This specific L7Ae sequence is utilized by Wang et al. in the development of a dual-regulation system for mammalian gene expression, in which L7Ae is paired with transcriptional repressor lacI (link to our LacI) to minimize leaky expression (Wang et al., 2023). Note that the original design of this L7Ae by Wang et al. included an HA tag (BBa_K1150016) at the 3’ end.

Usage

L7Ae can be utilized to repress certain genes, thus manipulating their expression. One may elect to add regulatory sequences in the 5’ UTR of L7Ae to allow L7Ae’s repression to be selective.

In the Micronaut gene circuit of the 2024 Stony Brook University iGEM Team, we built upon the advancements of Laird et al. and Wang et al. by implementing enhanced LacI (our LacI part link here), which was developed by Laird et al. (Lee et al., 2017), and L7Ae, 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 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, the other protein in our system, 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. 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 (Shu et al., 2023). 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 LacI and Argonaute2-based regulation, measured through relative GFP expression. We gathered multiple measures of the effectiveness of LacI and L7Ae, including different means of regulating LacI and L7Ae expression with microRNA 326. The approaches we took were based on the nature of bacterial gene expression and is discussed further on our wiki. We planned to gather data in these three approaches:

• A target site in the open reading frame, which is followed by the rest of the LacI-P2A-L7Ae coding sequence.
• A target site in the open reading frame which is followed by a P2A (BBa_K5090003), and then the rest of the above coding sequence
• A partial target site following an excerpt of the Ribosome Binding Site (BBa_J61100), followed by the start codon and then the rest of the above coding sequence.

Our wet lab and dry lab characterizations reflect the extent to which we were able to characterize these scenarios.

Wet Lab

In Wet Lab, we hope to test whether or not L7Ae expression will limit the expression of GFP. Our base-level GFP concentration in a cell free system is indicated in the graph below. Specifically at about 4 to 6 hours of expression using wild-type GFP in a cell-free system under Anderson promoter J23100 (BBa_J23100), we recorded nearly 300,000 RFU, with a plateau after 4 hours.

Figure 1: Graph representing relative fluorescence units (RFU) of cell-free system under different conditions, including negative and positive control.

Further testing revealed that expression of the dual-regulation system results in a statistically significant decrease in expression of GFP, although in this test its fluorescence was still within range of background by the empty pACYC backbone.

Figure 2: Graph demonstrating statistically significant decrease in GFP expression.

Further tests to confirm the dry lab predictions of L7Ae’s repression of this, discussed below, are ongoing.

Dry Lab

In Dry Lab, we characterized this L7Ae. We found that GFP expression was reduced to approximately 5.2*10^-8 M, from 2.5*10^-4 M. This demonstrates that this L7Ae is capable of significantly reducing expression of genes which have kink turns in advance of their coding sequence.

Figures 3 and 4: Graphs illustrating the effect of L7Ae upon GFP expression given GFP is placed under L7Ae kink-turn-based repression.

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 BglII site found at 358
  Illegal XhoI site found at 373
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

• Shu, W. J., Lee, K., Ma, Z., Tian, X., Kim, J. S., & 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.81818

• 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/gkx815

• Saito, H., Kobayashi, T., Hara, T., Fujita, Y., Hayashi, K., Furushima, R., et al. (2009). Synthetic translational regulation by an L7Ae–kink-turn RNP switch. *Nature Chemical Biology, 6*(1), 71–78. https://doi.org/10.1038/nchembio.273