Part:BBa_K5090001
Enhanced LacI
Description
LacI is a well studied transcriptional repressor that originated in E. coli. It achieves this transcriptional repression by binding to the lac operator (BBa_K079017) in a DNA sequence, preventing RNA polymerase from performing transcription of that DNA sequence into mRNA. This specific LacI sequence is utilized by Wang et al. in the development of a dual-regulation system for mammalian gene expression, in which LacI is paired with translational repressor L7Ae (BBa_K5090002) to minimize leaky expression (Wang et al., 2023). The sequence they used was originally developed by Laird et al. which in combination with two lac operators constituted a system that they state was greater than 100 times as strong as the wild type LacI (Laird et al., 2017). Additionally, it should be noted that IPTG, as per Kim et al., can bind to LacI and inhibit its binding activity to lac operators, thus freeing genes under lac operators to express when they would have otherwise been prevented from doing so by LacI (Kim et al., 2020). Finally, it should be noted that this sequence contains SV40 NLS, per the implementation by Wang et al.. This is not necessary for use in bacterial systems though also doesn’t necessarily have to be removed given bacteria simply lack nuclei, though is helpful for the mammalian implementations similar to those sought by the developers of this sequence.
Usage
LacI 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. Additionally, one may place the LacI gene itself under transcriptional or translational regulation.
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 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 (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 (BBa_K5090000), 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
Laird et al. and Wang et al. characterized the effectiveness of this enhanced LacI in their mammalian systems. Specifically, Laird et al. found their LacI to be 100 times stronger than the wild-type LacI (Laird et al., 2017), while Wang et al. found that this enhanced LacI limited leaky expression of firefly luciferase approximately 5%, whereas wild-type LacI still permitted approximately 35% of such leakage (Wang et al., 2023).
In our system, characterization was performed in combination with L7Ae 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 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 Anderson promoter J23100 (BBa_J23100), we recorded nearly 300,000 RFU, with a plateau after 4 hours. Tests to confirm the dry lab predictions of LacI’s repression of this, discussed below, are ongoing.
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 this enhanced LacI. We identified a hill coefficient for non-enhanced LacI from Krishna et al. (2013). Based on the affirmation of Laird et al. that enhanced LacI is capable of 100 times as strong repression, we modified the binding affinity accordingly. With this modification, we found that GFP expression was further reduced to 2.8*10^-6 M. This demonstrates that this LacI is capable of significantly reducing expression of genes which have lacO sites in advance of their coding sequence.
Figures 3 and 4: Graphs illustrating the effect of enhanced LacI upon GFP expression given GFP is placed under LacI.
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.
References
- 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
- Myung, S.-H., Park, J., Han, J.-H., & Kim, T.-H. (2020).
- 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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1081
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1081
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1081
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1081
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1081
- 1000COMPATIBLE WITH RFC[1000]
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