Parts
In our project, we registered four genetic parts.
This page provides a detailed explanation of each part, including its function and experimental evidence demonstrating its effectiveness.
| Part Number | Name | Type | Function |
|---|---|---|---|
| BBa_25Q0IEK8 | Cre recombinase for expression in(E.coli) | Cording | Excises, inverts, or relocates DNA sequences located between loxP sites. |
| BBa_2543OY9Q | Toluene o-xylene monooxygenase A113F mutant | Translational units | Expressed in E. coli, it oxidizes ethylene to ethylene oxide and can also oxidize aromatic compounds. |
| BBa_25DWKBRP | EtnR1/R2-controlled ethylene oxide sensor promoter | Regulatory | Induces expression of EtnR1 and EtnR2 upon IPTG stimulation, which activate the EtnP promoter in the presence of ethylene oxide. |
| BBa_25YQVUL6 | Cre-loxP NisinQ-to-fuGFP transcriptional switch | Generator | Cre recombinase excises the NisinQ domain via loxP sites, switching gene expression to GFP. |
Cre recombinase is an enzyme that catalyzes site-specific recombination between two identical loxP sequences, enabling genetic rearrangements such as DNA excision, inversion, and translocation. This part was designed to express Cre recombinase in Escherichia coli, and the expressed enzyme exhibited high recombination activity in both in vivo and in vitro systems. In our assay using the constructed loxP plasmid (BBa_25YQVUL6), clear DNA excision activity was observed.
The sequence design was based on pBbS5a-Opto-Cre-Vvd-2 (Nathan Tague et al., 2023)(1) and employs codons optimized for E. coli expression. The DNA fragment was obtained through synthetic gene synthesis by Twist Bioscience.
This part can serve as a core component for site-specific recombination control, applicable to genetic switching systems utilizing loxP sequences or condition-dependent gene expression modules.
This part encodes the A113F mutant of toluene o-xylene monooxygenase (TOM), a multicomponent monooxygenase enzyme complex derived from Burkholderia cepacia strain G4, which catalyzes the oxidation of ethylene into ethylene oxide. The TOM complex consists of six subunits (TomA0-A5), each contributing cooperatively to electron transfer and oxygen activation. Among them, TomA1, TomA3, and TomA4 form the catalytic core, assembling into a hexameric structure that facilitates molecular oxygen activation and substrate oxidation. TomA2 functions as a ferredoxin reductase, mediating electron transfer from NADH to the catalytic center, while TomA5 acts as an NADH oxidoreductase, supporting the electron donor system. The specific function of TomA0 remains incompletely understood.(2)
The A113F mutation replaces alanine at position 113 of the TomA3 subunit with phenylalanine, a modification known to broaden the oxidation specificity toward aromatic compounds and small hydrocarbons. This mutation is expected to enhance the oxidation efficiency of ethylene and promote ethylene oxide production by altering substrate selectivity.
The gene was designed based on the B. cepacia G4 sequence, introducing the A113F mutation and optimizing codon usage for expression in Escherichia coli. (Further details are provided in the Engineering section.) The DNA fragment was synthetically produced by Twist Bioscience. This enzyme system plays a key role in the conversion of ethylene to ethylene oxide, and within our project, it serves as the core catalytic module of an ethylene-responsive sensor system, in which the TOM-mediated product formation is coupled with the EtnR1/R2 regulatory circuit to control luminescent and fluorescent outputs.
This part is a promoter module regulated by the two-component system EtnR1/R2, which controls transcriptional activity in response to ethylene oxide (Eto). In this system, EtnR2 functions as a sensor kinase that binds Eto and undergoes autophosphorylation, subsequently transferring the phosphate group to the response regulator EtnR1. The phosphorylated EtnR1 then becomes activated and binds to a specific upstream regulatory sequence within the Etn promoter region, thereby enhancing the transcription of downstream genes. Through this mechanism, the cell can dynamically regulate gene expression in response to the presence of the exogenous chemical signal, ethylene oxide (3).
The design of this module was inspired by the construct reported by Claudia F. et al. (2023) (4), in which EtnR1, EtnR2, and EtnP were arranged on a single plasmid. Building upon this concept, we designed a single-plasmid configuration linking the expression regions of EtnR1/R2 with the EtnP promoter to improve the response efficiency of the sensor. The promoter region was derived from BBa_J435350 (a lac promoter-based sequence) included in the iGEM 2025 Distribution Kit, and the genetic module was assembled using the Golden Gate cloning method. The DNA sequence was synthetically generated by Twist Bioscience.
This system serves as a central component of our ethylene oxidation-based biosensor. The ethylene oxide produced through the oxidation of ethylene by toluene/o-xylene monooxygenase (TOM) acts as the input signal, which is sensed by EtnR2. The activated EtnR1 then induces transcription through the EtnP promoter, leading to the expression of a fluorescent reporter. Consequently, this module enables highly sensitive gene expression responses dependent on external chemical stimuli, functioning as the core signal transduction element in our biosensor design that couples chemical conversion to transcriptional activation.
This part is a gene switch module that enables Cre recombinase-dependent control of transcriptional output. It consists of a NisinQ-T7 terminator domain flanked by loxP sequences and a downstream Superfolder GFP (sfGFP)-T7 terminator domain. Upon expression of Cre recombinase, site-specific excision between the loxP sites is induced, removing the upstream NisinQ expression region and thereby switching transcriptional output from NisinQ to sfGFP. This configuration allows for an ON/OFF-type fluorescent output conversion depending on the presence or absence of Cre recombinase activity.
The part is placed under a common IPTG-inducible promoter, allowing reversible switching of transcriptional output upon Cre induction. In assay experiments using the Cre recombinase expression plasmid (BBa_25Q0IEK8), precise excision at the loxP sites and the corresponding shift in gene expression were clearly confirmed. Additionally, an upstream NusA expression region was incorporated to enhance the solubility of the expressed protein, thereby supporting stable NisinQ production.
The NisinQ expression unit was designed based on the NusA-NisQ-His construct (BBa_K4437002) reported by the Calgary iGEM Team (2022). The loxP sequences and downstream Cre-dependent recombination region were adapted from pBbS5a-Opto-Cre-Vvd-2 (Nathan Tague et al., 2023). To reduce synthesis complexity, partial sequence optimization was performed, and the final construct was assembled using the Gibson Assembly method. The DNA fragment was synthetically produced by Twist Bioscience.
This module serves as a fundamental unit for programmable transcriptional regulation circuits utilizing the Cre-loxP system. By integrating dual control via an external inducer (IPTG) and enzyme-mediated DNA excision, it enables highly precise switching of gene expression output. Furthermore, by employing sfGFP as a fluorescence reporter, the system allows quantitative visualization of Cre activity, making it applicable for analyzing the behavior of complex synthetic gene networks and for designing condition-responsive genetic devices.
This part can be safely and stably used under standard P1 (iGEM BSL1) experimental conditions.
To verify its expected function as a site-specific recombinase, a co-expression assay was performed using a reporter plasmid containing loxP sequences. In this experiment, the in vivo recombination activity of Cre recombinase was evaluated by co-transforming E. coli cells with a Cre expression plasmid and a switch plasmid harboring loxP sequences, followed by IPTG-induced expression.
The host strain used was the standard expression host E. coli BL21(DE3), which exhibited sufficient protein expression and enzymatic activity without requiring specialized rare-codon supplementation strains (e.g., Rosetta2). Cre gene expression was induced using IPTG concentrations ranging from 0 to 0.4 mM, and for each condition, the fluorescence intensity of GFP—expressed only after Cre-mediated DNA excision—was measured.(Fig1)
【Result】
We adopted data obtained from samples with OD600=0.5, and applied PBS as the blank.(Fig.2)
We performed Tukey's multiple comparison test to examine the fluorescence values.
Fluorescence intensity significantly increased upon IPTG induction compared to 0 mM (p < 0.05). The maximum expression was observed at 0.1 mM IPTG, while higher concentrations (0.2 mM and 0.4 mM) showed decreased fluorescence.
During liquid culture following transformation, this part was observed to cause a reduction in cell growth rate, although colonies formed normally on agar plates, suggesting that the metabolic burden may vary depending on culture conditions. The likely causes include leaky expression and plasmid load, in addition to the inherent structural complexity of the TOM complex. Furthermore, since toluene/o-xylene monooxygenase (TOM) is a multicomponent enzyme composed of several subunits, it was also confirmed that achieving stable assembly of the full enzyme complex within E. coli is not trivial.
No special biosafety requirements are associated with the use of this part, and it can be safely handled in a BSL1-level laboratory. When employing a T7 promoter system, it is advisable to use host strains that minimize leaky expression—particularly suppression strains such as E. coli BL21(DE3)pLysS are recommended for improved control.
For functional validation, induction experiments were performed using varying IPTG concentrations to confirm the expression of the Tom protein. Samples collected before and after induction were analyzed by SDS-PAGE, and the comparison of band intensities confirmed IPTG-dependent expression behavior of the TOM enzyme.(Fig.3)
【Result】
TomA0 = 8.3 kDa
TomA1 = 37.5 kDa
TomA2 = 10 kDa
TomA3 = 61.0 kDa
TomA4 = 13.1 kDa
TomA5 = 39.2 kDa
We observed several protein bands, but it was difficult to determine which ones corresponded to each Tom subunit, as the gel showed too many overlapping bands to distinguish clearly. In hindsight, tagging each protein would have allowed us to identify the subunits more reliably.
Although there were no significant issues in obtaining or using this part, it was observed that a portion of the expressed proteins became insoluble. In particular, EtnR2 exhibited low solubility within the cells and tended to accumulate as an insoluble fraction. Possible causes include a low codon adaptation index (CAI) and improper post-translational folding, both of which may interfere with the formation of the correct tertiary structure. While EtnR1 showed relatively stable expression, the efficiency of complex formation between EtnR1 and EtnR2 remained limited.
This part can be safely handled in a BSL1-level laboratory, although special caution is required when performing induction experiments using ethylene oxide (Eto) due to its volatility and toxicity. Eto volatilizes readily at approximately 10 °C and undergoes hydrolytic ring opening in aqueous environments; therefore, it should be dissolved in DMSO and handled exclusively within a fume hood.
For experimental validation, E. coli cells were transformed with the EtnR1/R2 expression plasmid, and protein expression was induced with IPTG. Post-induction samples were collected and analyzed by SDS-PAGE to confirm the expression of EtnR1 and EtnR2. (Fig.6)Considering the instability of Eto in aqueous media, the EtnR1/R2 expression was pre-induced prior to exposure, and cultures were subsequently supplemented with varying Eto concentrations to observe the response of the EtnR1/R2-EtnP system. Activation of the EtnP promoter resulted in GFP expression, and fluorescence intensity was used as a quantitative indicator to verify sensor responsiveness.(Fig.7)
【SDSPAGE】
The calculated molecular weights obtained using the ExPASy ProtParam tool were 63.3 kDa for EtnR1 and 21.8 kDa for EtnR2. In the soluble fraction, EtnR1 was detected slightly above the 66.2 kDa marker, whereas in the precipitated (insoluble) fraction, EtnR2 was observed above the 25 kDa marker. These results are consistent with the findings reported in the previous iGEM project “FRESH” (Sydney, Australia, 2016). However, the gel displayed multiple overlapping bands, making it difficult to clearly distinguish each protein. In retrospect, adding affinity tags to each subunit would have facilitated identification. Codon optimization should also be considered to improve the solubility and proper folding of EtnR2.
【Assay】
Despite UV light exposure, no detectable GFP expression was observed.
No particular issues were encountered in obtaining or using this part. It can be safely handled within a standard Escherichia coli expression system under BSL1 laboratory conditions. The use of E. coli BL21(DE3) is recommended, as this strain provided stable plasmid maintenance and reproducible expression results.
Functional validation was conducted under both in vitro and in vivo conditions. In the in vitro experiment, the site-specific recombination activity at the loxP sequences within this construct was evaluated using commercial Cre recombinase (NEB #M0298). The concentration of Cre enzyme was varied stepwise in the reaction system, and E. coli lysates were sampled over time for electrophoresis. A clear appearance of bands corresponding to the expected excision products was observed with increasing Cre concentrations, confirming that the designed site-specific recombination occurred as intended.(Fig.10)
The in vivo assay was performed under the same conditions as described for the Cre recombinase for expression in E. coli experiment.
【Result】
It was found that even when loxP_NisinQ_sfGFP is in a cyclized form, it can still undergo recombination under the control of Cre recombinase, with the efficiency of this process depending on both enzyme concentration and reaction time. This finding highlights that Cre recombinase is capable of acting on supercoiled plasmid DNA, which is an important and encouraging discovery.
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