Results

We used the replication origin (BBa_J435300) and lac promoter (BBa_J435350) included in the iGEM 2025 Distribution Kit as a basic template. First, the region containing the BsaI recognition sequence from the Kit plasmid was amplified by PCR and the EtnR1/R2 fragment was synthesised by IDT. These three fragments were ligated by Golden Gate Assembly (BsaI restriction enzyme), and the construction was confirmed by whole sequence analysis.

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Furthermore, Petn_fuGFP was introduced into this plasmid backbone via Gibson Assembly, and the full-length sequence was confirmed by analysis.

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The EtnR1R2_Petn_fuGFP_Gibson plasmid was then successfully constructed.

We conducted protein induction using IPTG and epoxyethane with the plasmid constructed in Section 1-1, but no GFP fluorescence was detected. Therefore, we performed SDS-PAGE to confirm whether EtnR1/R2 was being expressed properly.

First, induction was performed using IPTG (0.4 mM), after sonication of bacterial cells and the soluble and insoluble fractions were used as samples. For control samples, those prior to IPTG induction were similarly applied. The electrophoresis results are shown below.

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A band was observed for EtnR1 in the soluble fraction around 66 kDa. However, a strong band was detected in the insoluble fraction around 25 kDa for EtnR2.

These results suggest that EtnR2 is expressed but had folding abnormalities.

It was found that the incomplete folding of EtnR2 contributed to the dysfunction of the entire system. It was found that incomplete folding of EtnR2 affects the dysfunction of the entire expression system.

In this experiment, we constructed a Cre expression system utilizing the ethylene metabolic control mechanism and confirmed the expression of the transcription factors EtnR1/R2. A plasmid carrying an epoxyethane-responsive transcriptional control system (EtnR1R2-Petn-GFP) was successfully constructed by Golden Gate and Gibson Assembly. Furthermore, confirmation of transcription factor expression by SDS-PAGE suggested that EtnR1 was normally expressed, whereas EtnR2 was present in the insoluble fraction and may have no function. In the future, we have to improve the accuracy of ethylene response by advancing the improvement of EtnR2 folding and optimising expression conditions.

In this plasmid, expression was blocked by a terminator between the loxP sites before recombination, and GFP downstream was expressed after recombination. Also, NisinQ adopted the composite part with nusA (BBa_K4437002), considering its molecular weight and solubility in Escherichia coli.

We performed Gibson Assembly using a vector containing the downstream loxP sequence and GFP, and an insert containing the upstream loxP sequence, NusA_NisinQ, and a terminator. Full-length sequence analysis confirmed the successful construction of the loxP_NisinQ_sfGFP_Vector_Assembled plasmid.

We confirmed cleavage of the loxP site in the loxP plasmid (loxP_NisinQ_sfGFP_Vector_Assembled plasmid) with purified Cre recombinase. We used agarose gel electrophoresis to confirm the cleavage.

The results of the electrophoresis are shown in the figure below.

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From the electrophoresis results, distinct bands were observed in samples incubated with 4 units of Cre for 5 minutes and those incubated for 15 minutes. Thus, it was demonstrated that recombination occurs when the enzyme concentration is increased and incubation is short period.

To check for recombination at the DNA level in vivo, Cre plasmid and loxP plasmid were co-transformed into E. coli BL21 (DE3). We extracted plasmids from samples following IPTG induction, performed electrophoresis, and confirmed the function of Cre recombinase the cleavability of loxP in vivo. The results of the electrophoresis are shown in the figure below.

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Furthermore, we observed GFP fluorescence during bacterial cell recovery in the mini prep. The fluorescence is shown in the figure below.

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Electrophoresis results confirmed recombination of the loxP site by Cre under all conditions: IPTG 0/0.1/0.2/0.4 mM. The fact that cleavage was also observed in the absence of IPTG (-) suggests that Cre leakage expression was occurring.

We measured the fluorescence intensity of expressed GFP using a microplate reader and verified the effects of Cre leakage expression and IPTG concentration on GFP expression levels. E.coli having Cre and loxP plasmids were cultured overnight at 25°C with IPTG added at four concentrations: 0/0.1/0.2/0.4 mM (n=3). After protein induction, we took 1 mL of the culture medium and recovered the bacterial cells. Subsequently, we diluted the bacterial cells in PBS to OD600=0.5, applied this sample to a 96-well plate. Fluorescence at 535 nm was measured using excitation light at 485 nm. The measurement results are shown in the table and figure below.

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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. Although the value is low in the absence of IPTG, the presence of fluorescence suggests that leakage expression may be occurring.

This validation confirmed that the Cre/loxP system is functional both in vitro and in vivo. In vitro experiments demonstrated that reactions using purified Cre recombinase resulted in loxP site cleavage even after brief incubation periods. Furthermore, the reaction exhibited increased activity at high Cre concentrations. Also, the in vivo experiment confirmed GFP fluorescence in E. coli co-transformed with the Cre plasmid and loxP plasmid following IPTG induction, and electrophoresis further confirmed recombination of the loxP site under all conditions. These results show that the Cre/loxP system is also functional within cells. On the other hand, the fact that cleavage was observed even under conditions without IPTG addition suggested the possibility of Cre leakage expression. In the measurement of fluorescence intensity, the strongest fluorescence was observed at an IPTG concentration of 0.1 mM, and this concentration was found to be the optimal induction condition.

These results confirm that the Cre/loxP system functions as designed, and that the challenge for the future is to suppress leakage expression and establish a more stable control system.

TOM_TomA0124_Vector and TOM_A113F_Tom34_BsaI were ligated using Golden Gate Assembly. Agarose gel electrophoresis was performed to confirm the success of the Golden Gate Assembly. The electrophoresis results are shown in the figure below.

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Using the constructed TOM_A113F_GoldenGate plasmid, we verified whether TomA0-A5 expressed normally. First, E. coli cells induced with IPTG (A: 0.2 mM or B: 0.4 mM) were lysed, and both the soluble and insoluble fractions were applied. A sample from before IPTG induction was also run for comparison.

The theoretical molecular weights for each subunit are as follows:

TomA0 = 8.3 kDa, TomA1 = 37.5 kDa, TomA2 = 10 kDa, TomA3 = 61.0 kDa, TomA4 = 13.1 kDa, TomA5 = 39.2 kDa.

The electrophoresis results are shown below.

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We identified several bands, but we cannot be completely certain which Tom they correspond to. There were too many bands to confirm clearly. Therefore, the Dry group decided to perform simulations of Tom to confirm its function.

The Dry group's simulation results are here.

We successfully constructed a plasmid expressing Tom's A113F mutation and performed expression analysis. Although clear expression confirmation via SDS-PAGE was not achieved, simulations by the Dry group enabled theoretical evaluation of the mutation's structural significance and enzymatic function. Moving forward, we will refine expression conditions and culture environments based on the Dry group's simulation results, aiming to confirm stable expression and activation of Tom.