Part:BBa_K5090007
Dual Regulation System for E. coli-Based Systems
Description
The 2024 Stony Brook iGEM Team has developed a composite part consisting of an Anderson promoter (BBa_J23100), an Anderson ribosome binding site (BBa_J61100), a microRNA-326 target site (BBa_K5090006) followed by P2A (BBa_K5090004), enhanced LacI (BBa_K5090001), P2A (BBa_K5090005), L7Ae (BBa_K5090002), and the T1 terminator from E. coli rrnB (BBa_B0010). The portion from microRNA-326 followed by P2A through to the end of L7Ae is a continuous coding sequence, and this is achieved through use of the “self-cleaving peptide” P2A. This part is meant to create a “dual regulation system” in a manner similar to that achieved by Wang et al. (2023). This part is distinct from their implementation in two ways, both involving adaptation for use in a bacterial context.
This part utilizes an E. coli compatible promoter, ribosome binding site, and terminator, rather than the mammalian equivalents utilized by Wang et al. This part has a microRNA complementary target site in the open reading frame prior to LacI and L7Ae, meaning seven amino acids are coded for that are not meant to be part of any protein. To prevent this scar from impacting LacI, P2A is placed immediately following this site. The site was placed there as E. coli are best able to successfully express genes if start codons for those genes are maximum 7 nucleotides upstream of the Shine-Dalgarno sequence (“Help:Ribosome Binding Site - parts.igem.org”, 2024). As the miRNA target site is 20 nucleotides, placing this in the 5’UTR as Wang et al. did would be inadvisable in a bacterial implementation. To minimize potential issues and to make sure the addition of a 20 nucleotide scar onto P2A wouldn’t throw off the ORF, we added an additional T at the beginning of the target site, resulting in coding for serine first and restoring the open reading frame for the rest of the construct.
Usage
This bacterial dual regulation system can be utilized to selectively repress certain genes, thus manipulating their expression. As mentioned above, IPTG can prevent LacI from completing its repression by binding to LacI and prohibiting it from binding to lac operators. This construct’s coding sequence is already under the regulation of microRNA-326 and Ago2 (discussed further below), and one may modify this part to place the sequence under transcriptional or additional/different translational regulation.
In the Micronaut gene circuit of the 2024 Stony Brook University iGEM Team, we built upon the advancements of Laird et al. (2017) and Wang et al. by implementing enhanced LacI 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 (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), 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.
As stated above, this combined LacI-P2A-L7Ae coding sequence is preceded by a target complementary site for microRNA-326 and P2A. 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 at 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 hope to test whether or not LacI 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 an Anderson promoter, 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.
Additional testing is ongoing to confirm the dry lab predictions which are discussed below.
Dry Lab
In Dry Lab, we characterized the repression of GFP achieved by the enhanced LacI, courtesy of Laird et al., and L7Ae. Enhanced LacI and L7Ae repressed GFP by different amounts. With these two transcriptional and translational regulators both repressing the same GFP, expression was further reduced than either achieved alone. Finally, it is shown that Ago2 and miRNA represses the dual regulation system, thus allowing GFP to express again. This demonstrates that this dual-regulation system is capable of significantly reducing expression of genes which have lacO sites and kink turns in advance of their coding sequence, and more so than either repressor alone.
Figures 3, 4, 5, and 6: Graphs illustrating the effect of the dual repression system upon GFP expression given GFP is placed under LacI, as well as their own regulation by miRNA-326 and Ago2.
Note<b>
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.
<b>Sequence and Features</b>
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1214
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1214
Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1214
Illegal BglII site found at 1664
Illegal XhoI site found at 1679 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1214
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1214
- 1000COMPATIBLE WITH RFC[1000]
- 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
- 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
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