Parts
We designed and assembled a collection of modular genetic parts enabling the construction of toehold switch–based expression systems.
Our library includes transcriptional units, regulatory elements, and reporter constructs optimized for mammalian expression.
All parts were standardized, verified by sequencing, and deposited in the iGEM Registry to support future teams working on RNA-based regulation.
Our Parts Collection
Our genetic parts collection provides a comprehensive platform for selective gene expression in hepatocellular carcinoma cells. This enables the production of proteins with therapeutic potential exclusively in tumor-transformed cells, without affecting the function of healthy cells. It represents a key component in the context of anticancer therapy. The system is based on a short, designed RNA sequence located within the 5' untranslated region (5' UTR) of the transcript. Under physiological conditions, this sequence forms a secondary structure that prevents access to the translation initiation site. This structure acts as a receptor specific to the alpha-fetoprotein transcript - a marker characteristic for hepatocellular carcinoma cells. In the presence of this marker, the RNA hairpin unfolds, allowing translation to occur and thereby enabling the production of the target protein. The efficiency of the developed system is presented in the Results section.
Functional elements
The table includes genetic elements and constructs designed for the production of the final product - a functional transcriptional unit containing the selected toehold switch. It presents individual components as well as the complete vector intended for the cloning of specific toehold switches. The basic elements comprise sequences encoding fluorescent reporter proteins, which are used for direct testing of toehold switch specificity, as well as the sequence of the α-peptide of β-galactosidase (LacZ), enabling blue-white screening.
| Part Name | ID | Description |
|---|---|---|
| Mammalian optimized eGFP | BBa_25N3AWWS | Encodes eGFP protein, which emits green fluorescence when being excited with wavelengths between 450-490 nm. |
| Mammalian constitutive RFP reporter | BBa_25VE60AA | Encodes full mammalian transcription unit, that lead to constitutive high expression of RFP in transfected mammalian cells. |
| TU1 with cloning site for 5' UTR and CDS | BBa_25SH329F | Encodes transcription unit containing CMV promoter, strong 3' UTR and rabbit β-globin poly(A) signal. It contains cloning site for inserting 5' UTR and CDS using BsaI. Designed to be used to measure the translation under designed 5' UTR in mammalian cells. |
| TU1 with cloning site for 5' UTR and CDS - LacZ | BBa_254JMBEU | Encodes transcription unit containing CMV promoter, strong 3' UTR and rabbit β-globin poly(A) signal. It contains cloning site containing LacZ cassette for inserting 5' UTR and CDS and white-blue screening of colonies. Designed to be used to measure the translation under designed 5' UTR in mammalian cells. |
| Toehold switch testing cassette | BBa_25V2E0HW | Encodes a cassette for testing toehold switch activity in mammalian cells. Contain two transcription units - one with the cloning site for inserting toehold switch with the reporter protein of choice and another constitutively expressing RFP, as a control of transfection. |
| Backbone for assembly of 2 transcription units | BBa_259T9MVI | This is a vector providing backbone for Golden Gate Assembly of 2 transcription units. Contains lacZ cassette allowing white-blue colony screening with LacZ and IPTG and chloramphenicol resistance gene. |
| LacZ cassette | BBa_25DTJWTR | Encodes cassette expressing LacZ gene in the presence of IPTG in E. coli, what results in blue color of the colonies grown in the presence of X-Gal derived from BBa_J433047. Modified with overhangs allowing direct cloning of it into BBa_25LFVVO5. |
Toehold switches
Table presents the sequences of twenty-one designed toehold switches, each engineered for the selective binding of the alpha-fetoprotein (AFP) transcript. Under physiological conditions, the transcripts of these sequences adopt a secondary structure that, upon interaction with the cognate agonist, unfolds to expose the Kozak sequence, thereby enabling translation initiation.
| Part Name | ID | Description |
|---|---|---|
| AFP sensing toehold switch 1 | BBa_25ZHOR52 | Encodes toehold switch, designed to form secondary structure, which blocks translation (OFF state). In the presence of AFP mRNA, it binds to the toehold switch and unwinds it, leading to the release of start codon and initiation of translation (ON state). |
| AFP sensing toehold switch 2 | BBa_250CZ1N4 | |
| AFP sensing toehold switch 3 | BBa_25CBV2KZ | |
| AFP sensing toehold switch 4 | BBa_253W34O5 | |
| AFP sensing toehold switch 5 | BBa_258PVAE5 | |
| AFP sensing toehold switch 6 | BBa_25LTGU9W | |
| AFP sensing toehold switch 7 | BBa_25KMR72M | |
| AFP sensing toehold switch 8 | BBa_25EK59Z3 | |
| AFP sensing toehold switch 9 | BBa_2536A8PW | |
| AFP sensing toehold switch 10 | BBa_252Z2H1Q | |
| AFP sensing toehold switch 11 | BBa_25ZTWO4B | |
| AFP sensing toehold switch 12 | BBa_25MXPZ2S | |
| AFP sensing toehold switch 13 | BBa_257KS0RB | |
| AFP sensing toehold switch 14 | BBa_25RHES30 | |
| AFP sensing toehold switch 15 | BBa_25Q1KWBC | |
| AFP sensing toehold switch 16 | BBa_25OJUJ4Z | |
| AFP sensing toehold switch 17 | BBa_25LKZVOU | |
| AFP sensing toehold switch 18 | BBa_250L4JGE | |
| AFP sensing toehold switch 19 | BBa_25QCCD17 | |
| AFP sensing toehold switch 20 | BBa_25ZKTTE9 | |
| AFP sensing toehold switch 21 | BBa_25W42RKX |
eGFP elements
The table presents toehold switch sequences associated with the coding region of the enhanced Green Fluorescent Protein (eGFP), whose product serves as a reporter protein for the direct evaluation of toehold switch performance. Under physiological conditions, toehold switches adopt secondary structures that prevent the expression of eGFP. Upon the appearance of an agonist in the form of an alpha-fetoprotein (AFP) transcript, the hairpin structure unfolds, thereby enabling the initiation of eGFP translation.
| Part Name | ID | Description |
|---|---|---|
| AFP sensing toehold switch 1 +eGFP | BBa_255KXS8E | Encodes toehold switch, designed to form secondary structure, which blocks translation of eGFP (OFF state). When AFP mRNA is present, it binds to the toehold switch and unwinds it, leading to the release of start codon and initiation of translation of eGFP (ON state). |
| AFP sensing toehold switch 2 +eGFP | BBa_25OHWS9U | |
| AFP sensing toehold switch 3 +eGFP | BBa_25P40TUI | |
| AFP sensing toehold switch 4 +eGFP | BBa_25WKB8EQ | |
| AFP sensing toehold switch 5 +eGFP | BBa_25DE4PM1 | |
| AFP sensing toehold switch 6 +eGFP | BBa_25LLAQMP | |
| AFP sensing toehold switch 7 +eGFP | BBa_258EGC7I | |
| AFP sensing toehold switch 8 +eGFP | BBa_25M4C8KD | |
| AFP sensing toehold switch 9 +eGFP | BBa_25JN4KH4 | |
| AFP sensing toehold switch 10 +eGFP | BBa_25CAXBKO | |
| AFP sensing toehold switch 11 +eGFP | BBa_25LVR6GD | |
| AFP sensing toehold switch 12 +eGFP | BBa_25T247XK | |
| AFP sensing toehold switch 13 +eGFP | BBa_25C53YTI | |
| AFP sensing toehold switch 14 +eGFP | BBa_256TOU7T | |
| AFP sensing toehold switch 15 +eGFP | BBa_25X549TG | |
| AFP sensing toehold switch 16 +eGFP | BBa_25XWVEXZ | |
| AFP sensing toehold switch 17 +eGFP | BBa_25W6KU7P | |
| AFP sensing toehold switch 18 +eGFP | BBa_25KWUOMK | |
| AFP sensing toehold switch 19 +eGFP | BBa_25YFIIUQ | |
| AFP sensing toehold switch 20 +eGFP | BBa_25M17XDK | |
| AFP sensing toehold switch 21 +eGFP | BBa_25NVO2ZB | |
| 5UTR13 + eGFP | BBa_253UBWIF | Encodes eGFP under the control of strong 5' UTR. |
qPCR primers
The table presents primers used in qPCR reactions designed to evaluate the expression levels of alpha-fetoprotein (AFP) and gasdermin D (GSDMD) in the analyzed cell lines. AFP serves as a tumor marker and functions as an agonist for the designed toehold switches. Gasdermin D, on the other hand, is an effector protein that induces pyroptosis, with its expression regulated through a corresponding toehold switch. The table also includes primers for a reference gene, which was used to normalize and determine the relative expression levels of the analyzed genes.
| Part Name | ID | Description |
|---|---|---|
| AFP_F | BBa_25QPUUES | Forward primer designed to amplify the AFP gene. The primer is 20 nucleotides long, with a melting temperature (Tm) of approximately 58.0 °C and a GC content of 50.0%. It exhibits very low self-complementarity (2.0) and no 3′ self-complementarity (0.0), minimizing the risk of primer-dimer formation. This primer is expected to specifically anneal to the AFP target sequence under standard PCR conditions, ensuring efficient and reliable amplification of the gene fragment. |
| AFP_R | BBa_2527BQKZ | Reverse primer designed to amplify the AFP gene. The primer is 22 nucleotides long, with a melting temperature (Tm) of approximately 59.3 °C and a GC content of 50.0%. It demonstrates moderate self-complementarity (5.0) and low 3′ self-complementarity (1.0), minimizing the risk of primer-dimer formation and ensuring stable binding during amplification. This reverse primer is expected to specifically anneal to the complementary strand of the AFP target sequence under standard PCR conditions, enabling efficient and accurate amplification of the gene fragment in combination with the AFP forward primer. |
| GSDMD-NT_F | BBa_25GRWIBZ | The GSDMD-NT forward primer is designed to specifically anneal to the N-terminal region of the Gasdermin D (GSDMD) gene, enabling its efficient amplification in PCR-based assays. This region encodes the pore-forming domain responsible for pyroptosis activation. The primer’s optimal thermodynamic properties and low self-complementarity ensure high specificity and stable binding, making it suitable for use in gene expression studies, functional assays, and molecular analyses investigating the role of GSDMD in inflammatory cell death pathways. |
| GSDMD-NT_R | BBa_25CKF5M2 | The GSDMD-NT reverse primer is designed to specifically anneal to the complementary strand of the N-terminal region of the Gasdermin D (GSDMD) gene, enabling precise and efficient amplification of the target fragment in PCR assays. With optimal thermodynamic parameters and minimal self-complementarity, this primer ensures stable binding and reduces the risk of primer-dimer formation. When used together with the corresponding forward primer, it allows accurate detection and quantification of GSDMD-NT expression, supporting studies focused on pyroptosis, inflammatory signaling, and cell death mechanisms at the molecular level. |
| GAPDH_F | BBa_25GHP2EI | The GAPDH forward primer is designed to specifically bind to the housekeeping gene GAPDH, enabling stable and reproducible amplification of its mRNA (after reverse transcription) or genomic fragment in PCR assays. With a length of 21 nucleotides, a melting temperature (Tm) of about 59.65 °C, and a GC content of 47.62%, the primer ensures a strong yet balanced binding affinity to the target sequence. Its low self-complementarity score (3.0) and zero 3′ self-complementarity (0.0) minimize the risk of primer-dimer formation or internal secondary structure interference, particularly at the critical 3′ end. These favorable thermodynamic features help maintain efficient and specific annealing, making this primer suitable for quantitative real-time PCR (qRT-PCR) and gene expression normalization in diverse samples and experimental settings, as described in the referenced study. |
| GAPDH_R | BBa_25M1Z082 | The GAPDH reverse primer, reported in PMC9192978, anneals to the complementary strand of the GAPDH gene to enable accurate amplification in PCR and qRT-PCR assays. With a length of 21 nucleotides, Tm of 57.99 °C, and GC content of 47.62%, it ensures stable and efficient binding. Its low self-complementarity (3.0) and 3′ self-complementarity (2.0) reduce dimer formation, supporting reliable use in gene expression normalization and reference gene analysis. |
Cloning Primers
The table lists the primers used for the isolation of genetic elements, their modification with appropriate flanking sequences facilitating the cloning process, and for the verification of the resulting genetic constructs.
| Part Name | ID | Description |
|---|---|---|
| Colony_PCR_F | BBa_25YFS6TE | Forward primer for colony PCR binding to the part of rabbit β-globin poly(A) sequence. Used for colony PCR to check the presence of mammalian transcription units and if the insert is in them or not. |
| Colony_PCR_R | BBa_25CMDKAL | Reverse primer for colony PCR binding to the part of CMV promoter sequence. Used for colony PCR to check the presence of mammalian transcription units and if the insert is in them or not. |
| RFPseq | BBa_25JMQ293 | Primer for sequencing and colony PCR specific to RFP containing transcription unit. Tested on the colonies with transcription unit with RFP. |
| LacZ_F | BBa_2586BTCP | This primer was designed to amplify lacZ cassette, used for blue-white screening of E. coli colonies, from plasmid BBa_259T9MVI. It contains overhang allowing PCR product to be cloned into BBa_252RGHOZ using BsaI digestion and ligation. |
| LacZ_R | BBa_25928ERZ | This primer was designed to amplify lacZ cassette, used for blue-white screening of E. coli colonies, from plasmid BBa_259T9MVI. It contains overhang allowing PCR product to be cloned into BBa_252RGHOZ using BsaI digestion and ligation. |
| TU2_F | BBa_258AF7WU | Primer specific to the beginning of BBa_25VE60AA allowing amplification of it. |
| TU2_R | BBa_252JUY03 | Primer specific to the end of BBa_25VE60AA allowing amplification of it. |
| TU2_OV_F | BBa_256US3FC | Primer to amplify pTwist Amp High Copy vector with 20-nt overhangs on the ends, complementary to the beginning of BBa_25VE60AA. This allows for it to serve as a destination vector for BBa_25VE60AA during HiFi cloning. |
| TU2_OV_R | BBa_252XEY4S | Primer to amplify pTwist Amp High Copy vector with 20-nt overhang on the ends, complementary to the end of BBa_25VE60AA. This allows for it to serve as a destination vector for BBa_25VE60AA during HiFi cloning. |
Transcriptional units incorporating toehold switches positioned upstream of eGFP
The table presents functional transcriptional units incorporating toehold switches positioned upstream of eGFP (BBa_25N3AWWS). Each construct also includes a promoter sequence (BBa_J433000), a 3' UTR (BBa_J433018), and a poly(A) sequence (BBa_J433023). These constructs constitute a ready-to-use platform for eGFP expression regulated by the specific characteristics of the toehold switch.
| Part Name | ID | Description |
|---|---|---|
| AFP sensing toehold switch eGFP TU 1 | BBa_25AFWI65 | Encodes a toehold switch–containing transcriptional unit designed to regulate eGFP translation in response to AFP mRNA. The construct forms a secondary structure that sequesters the ribosome binding site and start codon, keeping translation OFF under normal conditions. When AFP mRNA is present, it hybridizes with the trigger region of the toehold switch, unfolding the structure and exposing the ribosome binding site. This conformational change allows translation initiation and eGFP production (ON state). Such RNA-based regulatory elements enable programmable sensing of AFP transcripts and can serve as diagnostic biosensors for biomarker detection. |
| AFP sensing toehold switch eGFP TU 2 | BBa_258INMBJ | |
| AFP sensing toehold switch eGFP TU 3 | BBa_253ILJRI | |
| AFP sensing toehold switch eGFP TU 4 | BBa_250VNI4E | |
| AFP sensing toehold switch eGFP TU 5 | BBa_259WW1OO | |
| AFP sensing toehold switch eGFP TU 6 | BBa_25PU5Q27 | |
| AFP sensing toehold switch eGFP TU 7 | BBa_25X1NIS4 | |
| AFP sensing toehold switch eGFP TU 8 | BBa_2584PTI1 | |
| AFP sensing toehold switch eGFP TU 9 | BBa_252V0MM7 | |
| AFP sensing toehold switch eGFP TU 10 | BBa_25GC8TPI | |
| AFP sensing toehold switch eGFP TU 11 | BBa_25LY4UR8 | |
| AFP sensing toehold switch eGFP TU 12 | BBa_25ESVXUM | |
| AFP sensing toehold switch eGFP TU 13 | BBa_25X4CT6Z | |
| AFP sensing toehold switch eGFP TU 14 | BBa_25W6GSVV | |
| AFP sensing toehold switch eGFP TU 15 | BBa_25O0IG16 | |
| AFP sensing toehold switch eGFP TU 16 | BBa_25UN9KGQ | |
| AFP sensing toehold switch eGFP TU 17 | BBa_250PAGEP | |
| AFP sensing toehold switch eGFP TU 18 | BBa_25W81Z1F | |
| AFP sensing toehold switch eGFP TU 19 | BBa_250I00Z9 | |
| AFP sensing toehold switch eGFP TU 20 | BBa_25OE6E8R | |
| AFP sensing toehold switch eGFP TU 21 | BBa_254JLP5R |