The system we developed was based on a modular architecture consisting of three main plasmids — reporter, sensor, and expression — each performing a specific function within a single regulatory framework. All plasmids were constructed using standard BioBrick elements combined with our own engineered components, making the system compatible with other iGEM parts and adaptable for use in related synthetic biology tasks.
The reporter plasmid contained the GFP gene under the control of the pTet promoter, allowing visualization of the regulatory response.
The sensor plasmid carried the TetR repressor and dCas proteins under constitutive promoters of varying strengths, which made it possible to adjust the level of transcriptional repression and evaluate promoter-dependent activity.
The expression plasmid was responsible for the production of guide RNAs (sgRNAs), including both standard and modified versions designed to form RNA–RNA interactions through kissing-loop structures. The control variant of this plasmid (P3sgRNAgen) contained an unmodified sgRNA scaffold and was used as a reference for evaluating the activity of hairpin-containing constructs.
The modular combination of these three plasmids allowed us to fine-tune the transcriptional activity of the TetR/pTet regulatory system and evaluate the stability and specificity of RNA–RNA interactions in the context of dCas-based complexes. This structure also made it possible to create new variants of inducible circuits and selection systems — for example, a chloramphenicol-resistance module under the control of the pTet promoter, which can be used both for clone selection and for studying the efficiency of TetR repression.
All plasmids were tested in E. coli Top10 cells and showed correct operation of their regulatory elements, confirming the functionality and flexibility of the modular design.
All parts listed below were used to assemble the reporter, sensor, and expression plasmids described in our experiments.
These parts were taken from the iGEM Registry and used as building blocks for the regulatory modules in our constructs.
| Component | Part ID | Well | Antibiotic | Plasmid Size (bp) | Target Size (bp) |
|---|---|---|---|---|---|
| Constitutive promoter | BBa_J23114 | I5 | Chloramphenicol | 2424 | 39 |
| Strong constitutive promoter | BBa_J23110 | G5 | Chloramphenicol | 2424 | 39 |
| Weak constitutive promoter | BBa_J23116 | K5 | Chloramphenicol | 2424 | 39 |
| RBS (Ribosome Binding Site) | BBa_J428032 | O5 | Chloramphenicol | 2413 | 28 |
| TetR gene (repressor) | BBa_J428028 | M4 | Chloramphenicol | 2670 | 625 |
| Terminator 1 | BBa_J428092 | C1 | Chloramphenicol | 2520 | 135 |
| Terminator 2 | BBa_J428091 | A1 | Chloramphenicol | 2432 | 47 |
These elements formed the foundation of our three-plasmid system. Promoters of varying strength enabled controlled TetR and dCas expression, while the RBS and terminators ensured efficient transcription and translation termination.
This subcollection represents the functional units used to assemble our composite inducible chloramphenicol-resistance system and the expression modules tested in the project.
| Part No. | Part Name | Description |
|---|---|---|
| BBa_2500KIWZ | Constitutive promoter with two targets around it gP3gP7R | Created based on the constitutive promoter from the BBa_J23116 collection. Modified by adding two target sequences (gP3 and gP7) for binding dCas9 proteins in the PAM orientation. The promoter is located between the two target sequences. The distance between the PAM sites is 125 bp, corresponding to the distance in mcccDNA. |
| BBa_25EFHPDN | Constitutive promoter with two targets before it gP3gP7B | Created based on the constitutive promoter from the BBa_J23116 collection. Modified by adding two target sequences (gP3 and gP7) for binding dCas9 proteins in the PAM orientation. The promoter is located after the pair of target sequences. The distance between the PAM sites is 125 bp, corresponding to the distance in mcccDNA. |
| BBa_25FVO7QU | Constitutive promoter with two targets after it gP3gP7A | Created based on the constitutive promoter from the BBa_J23116 collection. Modified by adding two target sequences (gP3 and gP7) for binding dCas9 proteins in the PAM orientation. The promoter is located before the pair of target sequences. The distance between the PAM sites is 125 bp, corresponding to the distance in mcccDNA. |
| BBa_25B74W45 | CRISPR-mediated DNA looping selection vector gP3gP7A | A composite part based on the BBa_J23116 promoter (in the gP3gP7A configuration) and the TetR protein (BBa_J428028). It is used as a genetic network element and is inversely inverted. The promoter is located before the pair of target sequences. The principle of action: a pair of Cas proteins forms a DNA loop in the promoter region, preventing TetR synthesis. Used to select the most stable interactions between two Cas proteins. |
| BBa_25P3WVLT | CRISPR-mediated DNA looping selection vector pgP3gP7B | A composite part based on the BBa_J23116 promoter (in the gP3gP7B configuration) and the TetR protein (BBa_J428028). It is used as a genetic network element and is inversely inverted. The promoter is located after the pair of target sequences. The principle of action: a pair of Cas proteins forms a DNA loop in the promoter region, preventing TetR synthesis. Used to select the most stable interactions between two Cas proteins. |
| BBa_25QL9PUD | CRISPR-mediated DNA looping selection vector pgP3gP7R | A composite part based on the BBa_J23116 promoter (in the gP3gP7R configuration) and the TetR protein (BBa_J428028). It is used as a genetic network element and is inversely inverted. The promoter is located between the pair of target sequences. The principle of action: a pair of Cas proteins forms a DNA loop in the promoter region, preventing TetR synthesis. Used to select the most stable interactions between two Cas proteins. |
| BBa_25UCIH90 | pTet_sfGFP for DNA looping selection | A reporter plasmid expressing the sfGFP protein under the control of the pTet promoter. It is part of a selection system where reverse inverters act as the TetR protein. In the absence of DNA loop formation on the regulatory plasmid (which expresses TetR), expression of the sfGFP reporter protein from this plasmid does not occur. |
| BBa_2543441S | pTet_CmR for DNA looping selection | A reporter plasmid expressing the chloramphenicol resistance gene (CmR) under the control of the pTet promoter. It is part of a selection system where reverse inverters act as the TetR protein. In the absence of DNA loop formation on the regulatory plasmid (which expresses TetR), expression of the CmR resistance gene from this plasmid does not occur. |
Together, these parts formed the basis for constructing and testing the reporter, sensor, and expression plasmids, including the inducible CmR composite part.