Fig1. Confirmation of the transformation efficiency of the prepared EcN competent cells.

8/19-8/20: The successful transformation of EcN has been confirmed (acquisition of Cm resistance). Now, the transformation experiment with the created vector will begin.

Using TWIST Bioscience's DNA synthesis service, two DNA fragments were designed and synthesized.

The fragments were amplified through PCR, removing the adapters in the process.

The backbone was obtained by PCR from the iGEM distribution kit. Complementary primers were designed to introduce a 20 bp overlap sequence (matching the primer sequence) between each fragment.

The vector was assembled using the NEBuilder® HiFi DNA Assembly Cloning Kit (NEBuilder).

PCR of the fragments, electrophoresis, and purification from the gel.

Fig2.Agalose Gel Extraction of K4387006-fragment 1, 2

Since the polymerase we used (TaKaRa Ex Premier DNA Polymerase) cannot calculate the annealing temperature, we performed PCR by setting the temperature to a gradient.

Fig3. Agalose Gel Extracton of K4387006-gragmant2, Backbone

Similarly, we performed gradient PCR and amplified the backbone in a linear form. The gel purification of K4387006-fragment2 failed, so we performed PCR again.

Fig4. Results of DNA Fragment Extraction from Agarose Gel

We measured the concentration of each purified fragment using NanoDrop 1500. This concentration is referenced for the amount added to NEBuilder.

Assembly of Vector (PnorV-sfGFP-norR) Using NEBuilder and Transformation of EcN

Three DNA fragments with overlapping sequences were assembled using NEBuilder.

Fig 5. Vector map pSB1C3(pNorV-sfGFP-norR)

We confirmed whether the vector assembly was successful by performing a transformation.

Fig 6. Transformation of EcN with Vector (K4387006: pNorV-sfGFP-norR) and Cultivation

The successful assembly and transformation of the vector were confirmed by colony PCR (Insert check: VR primer and VF2 primer).

Fig 7. Colony PCR of the transformant EcN (pNorV-sfGFP-norR)

The amplification of the insert with a length of 3026 bp was successfully confirmed.

We measured the OD600 and fluorescence (Ex: 470 nm, Em: 510 nm) of E. coli Nissle 1917 (EcN), which was introduced with pNorV-sfGFP-norR (a nitric oxide-responsive promoter and sfGFP), at seven different NO concentration conditions (0 µM, 5 µM, 16.66 µM, 50 µM, 166.66 µM, 250 µM, 500 µM) using a plate reader at one-hour intervals.

For each condition, 3 colonies (colony PCR confirmed) and 3 samples per colony were measured.

The samples were prepared in a 96-well plate as shown below.

Table 1. Assay 1 96-well (Concentrations represent the amount of DETA/NONOate added)

0 µM	0 µM	0 µM	0 µM	0 µM	0 µM	0 µM	0 µM	0 µM
5 µM	5 µM	5 µM	5 µM	5 µM	5 µM	5 µM	5 µM	5 µM
16.66 µM	16.66 µM	16.66 µM	16.66 µM	16.66 µM	16.66 µM	16.66 µM	16.66 µM	16.66 µM
50 µM	50 µM	50 µM	50 µM	50 µM	50 µM	50 µM	50 µM	50 µM
166.66 µM	166.66 µM	166.66 µM	166.66 µM	166.66 µM	166.66 µM	166.66 µM	166.66 µM	166.66 µM
250 µm	250 µm	250 µm	250 µm	250 µm	250 µm	250 µm	250 µm	250 µm
500 µM	500 µM	500 µM	500 µM	500 µM	500 µM	500 µM	500 µM	500 µM
balnk 1	blank 2	balnk 3	-	-	-	-	-	-

It was discovered that there was contamination in the blank during the OD600 measurement one hour after the start.

As a result, we immediately proceeded with sample preparation for Assay 2.

We adjusted the samples immediately and began measurement because there was contamination in Assay 1.

We prepared the samples in a 96-well plate as shown below.

Table 2. Assay 2, 96-well Plate (Concentrations represent the amount of DETA/NONOate added)

0 µM	0 µM	0 µM	Blank 1
5 µM	5 µM	5 µM	Blank 2
16.66 µM	16.66 µM	16.66 µM	Balnk 3
50 µM	50 µM	50 µM
166.66 µM	166.66 µM	166.66 µM
250 µm	250 µm	250 µm
500 µM	500 µM	500 µM

Fig 8. Time-Dependent Response of the norV Promotor to DETA/NO

Fig9. Time-Dependent Response of the norV Promotor to DETA/NO

We were able to verify the reproducibility of the experiments conducted by UZurich-iGEM2022 regarding the functionality of the sensor.

By measuring the absorbance of DETA/NO at 252 nm and comparing it to the initial concentration, it may be possible to calculate the accurate NO concentration at each time point.

This step is necessary to align with the intestinal nitric oxide levels observed in patients.

The notation "X µM DETA/NO" represents the initial condition of DETA/NO, but the exact concentration of NO changes over time as it is time-dependent.

We mistakenly believed that DETA/NO would fully react, releasing all the NO in proportion to its molar amount, and that the concentration would gradually decrease over time. This misunderstanding led us to set these somewhat arbitrary initial DETA/NO concentrations. In reality, we intended to focus on the response at concentrations of 333.33 µM for patients and 33 µM for healthy individuals.

Yet another mistake has been discovered. According to a report by Zsuzsa Bebok and colleagues[1], DETA/NONOate releases between 450 nM and 350 nM of nitric oxide per 100 µM over 12 hours. Based on this, we decided to revise the graph to reflect these findings.

Fig 10. Fig 11. Prediction of NO emissions from DETA NONOate.

Additionally, we discovered a significant mistake in our calculations. (September 30, 2024)

The nitric oxide concentration in the intestines of patients, as reported by Lundberg JO, is 1,255 nM, not 333.33 µM.

Similarly, the nitric oxide concentration in healthy individuals is 7.3 nM, not 33.33 µM.

This error was caused by a unit conversion mistake on our part.

Thus, our measurement conditions are as follows:

DETA/NONOate: 0 µM	NO: 0 nM
DETA/NONOate: 5 µM	NO: 17.5~22.5 nM
DETA/NONOate: 16.66 µM	NO: 58.31~74.97 nM
DETA/NONOate: 50 µM	NO: 175~225 nM
DETA/NONOate: 500 µM	NO: 1750~2250 nM

Procedure Overview:

Pre-culture of EcN (pNorV-sfGFP):

1. Inoculated colonies into LB + chloramphenicol and incubated overnight at 37°C with shaking.

Preparation of Reaction Mixture:

2. Created mixtures by adding bacterial culture to LB, keeping it on ice.

3. Dispensed bacterial suspension and LB into wells of a 96-well plate.

4. Preparation of DETA/NONOate and H₂O₂:

5. Prepared required concentrations of DETA/NONOate and H₂O₂ for the assay.

6. Well Plate Setup:

7. Organized the wells into different treatments for DETA/NONOate and H₂O₂, including controls and combinations.

Measurement:

8. Incubated the plate at 37°C and measured fluorescence intensity (excitation at 470 nm, emission at 510 nm) and OD600 hourly using the plate reader.

Key Conditions for Plate Wells:

Various concentrations of DETA/NONOate and H₂O₂ were tested, with specific conditions outlined for each column in the 96-well plate.

Figure 12: Time-Dependent Response of the norV Promotor to DETA/NO, H2O2 or DETA/NO and H2O2

Fig 13.Time-Dependent Response of the norV Promotor to DETA/NO, H2O2 or DETA/NO and H2O2

Fig 14.Time-Dependent Response of the norV Promotor to DETA/NO, H2O2 or DETA/NO and H2O2

We create the vector: pSB1C3(K5349005).

We designed overlapping sequences for three fragments using NEBuilder, ordered the insert parts (two fragments) through TWIST's DNA synthesis service, and performed PCR for the backbone using the vector from the IGEM Distribution Kit as a template.

Linearization PCR of the backbone, amplification of K5349005 fragment 1 and 2 by PCR, and adapter removal.

The DNA synthesis service by TWIST Bioscience has adapter sequences added to both ends.

By performing PCR, amplification and removal of the adapters can be done simultaneously.

At this point, the primers will be designed to have overlapping sequences at both ends, ensuring that they are complementary to each other.

Fig15. Agalose Gel Extracton of UL23-fra1, 2(K5349005), Backbone

After confirming the success of the PCR, we purified the product from the gel and measured its concentration using NanoDrop.

Fig 16.17.Results of DNA Fragments Extraction from Agarose Gel

Based on the measured concentrations, we assembled using NEBuilder and transformed EcN.

Fig 18.Transformation of EcN with Vector (K5349005: UL23-sfGFP) and Cultivation

Since we obtained transformants, we checked the length of the insert using colony PCR.

Fig 19. Colony PCR of EcN (K5349005: UL23-sfGFP)

Since the insert length of K5349005 is 2362 bp, we decided to use colony 3 for future experiments.

Additionally, we will confirm the sequence of this vector through Sanger sequencing.

Objective:

The aim of this experiment is to assess the growth-inhibiting effects of the HSV-TK/GCV system on Escherichia coli (E. coli), with the goal of introducing it as a "kill switch" suicide gene. In this trial, GCV concentrations will be gradually increased while subculturing to determine if the experimental conditions are suitable for obtaining accurate data in future experiments.

Experimental Method:

1. E. coli was cultured in a shaking incubator at 37°C in LB medium supplemented with chloramphenicol (LB+Cm) until the optical density (OD600) reached 0.5.

2. The culture was then divided into two parts:

• One part served as the control.

• The other part was treated with GCV (Ganciclovir).

3. The OD600 of both cultures was measured every 15 minutes to monitor bacterial growth.

4. Since no significant growth inhibition was observed, the GCV concentration was progressively increased in the following steps:

• 1 µM → 1.6 µM → 2.0 µM → 10.0 µM → 100.0 µM.

Results:

We attempted to enhance GCV sensitivity by introducing the SR39 mutation into the UL23 gene (see Engineering Success for details).

We extracted pSB1C3-K5349005 using miniprep, and using this plasmid as a template, we performed Inverse PCR to introduce the SR39 mutation.

Fig 2O. Agarose Gel Extraction of Inverse PCR

Fig 21. Transformed colonies were obtained

Since E. coli cannot grow with vectors that have two origins of replication, if no mutations occurred during PCR, we can assume that the desired vector was successfully assembled.

The plasmid was extracted from the cells using miniprep and sent for Sanger sequencing.

Perform the assay with SR39-mutated HSV-TK.

Experimental conditions: Three samples of EcN (UL23 SR39) with equal initial turbidity were prepared. GCV (1.0 μM) was added to one sample, and the samples were shaken at 37°C and 180 rpm for 5.5 hours. OD660 was measured every 15 minutes.

Results

Fig 22. HSV-TK/GCV Assay2 (n=3)

No significant growth inhibitory effect was observed.

Verification of growth inhibition effect at GCV 100 μM. Since no significant difference was observed at GCV 1 μM, aliquots were taken from Sample 1-3 GCV- and sub-cultured. GCV was added to one of each sample to reach 100 μM, creating 3 GCV- and 3 GCV+ samples. (The initial turbidity was matched between samples with the same number.)

A total of 6 samples were shaken at 37°C and 180 rpm, and turbidity was measured after 8.5 hours.

Results

Fig 23.Assay 2(additional) GCV 100 µM (n=1)

Purpose:

The aim of this assay is to further evaluate the growth-inhibiting effects of the HSV-TK/GCV system using the SR39 mutation of the HSV-TK gene. This assay investigates both colony formation ability on GCV plates and the growth inhibition in liquid culture by measuring optical density (OD) and viable cell counts over time.

Methods:

1. Pre-culture and Sample Preparation:

Inoculate EcN (pSB1C3-UL23SR39) (TK+) and EcN (pSB1C3) (TK-) colonies in LB medium, measure OD600, and split the cultures into three aliquots each.

2. Colony Formation Inhibition on GCV Plates:

Plate TK+ and TK- samples on LB plates with GCV concentrations of 10 µM, 100 µM, and 1000 µM, and assess colony formation the next day.

3. Growth Inhibition by GCV (OD600 Measurement):

Add GCV (100 µM) to the six samples and measure OD600 every 30 minutes to monitor growth inhibition.

4. Measurement of Viable Cell Counts at Each Time Point After GCV Addition:

Take aliquots at 0, 2, 4, and 6 hours, perform serial dilutions, and plate the samples to assess viable cell counts.

Results:

Fig 24. HSV-TK/GCV Assay3 Colony formation inhibition on GCV plates (n=3)

Fig 25.HSV-TK/GCV Assay3 - Growth Inhibition by GCV (OD600 Measurement)

Fig 26. HSV-TK/GCV Assay3 - Measurement of Viable Cell Counts at Each Time Point After GCV Addition

Discussion:

In the comparison between TK- and TK+, the viable cell counts of TK+ were significantly higher across GCV concentrations ranging from 0 µM to 100 µM, while at 1000 µM GCV, the counts were similar. Within the TK+ comparison, it can be inferred that a proliferation suppression effect was observed between the GCV plate (1000 µM) and the GCV plates (0, 10, and 100 µM). However, there were several instances where colony counts could not be measured, resulting in an insufficient number of statistically reliable samples. Due to issues with experimental techniques and dilution concentration settings, it is necessary to conduct a re-experiment.

Purpose:

Assay 4 aims to investigate the effects of Ganciclovir (GCV) on the growth of TK- and TK+ strains in both solid and liquid media. The assay will assess viable cell counts on GCV plates and monitor turbidity and viable cell counts over time after GCV addition in liquid culture.

Methods:

1. Pre-culture of TK- and TK+:

•Prepare 6 mL cultures of both TK- and TK+ strains in LB medium.

•Pre-culture both strains overnight at 37°C with shaking (225 rpm).

•After pre-culture, standardize the turbidity (OD600) of the TK- and TK+ samples as much as possible by adding fresh LB medium to the cultures.

2. Viable Cell Count Measurement on GCV Plates:

• Take 10 µL from each of the 6 pre-cultured tubes and dilute it into 990 µL of LB medium.

• Perform this dilution step three times to achieve a final 10^6-fold dilution.

• Plate 100 µL of the diluted cultures onto GCV plates with three different GCV concentrations: 0 µM, 100 µM, and 1000 µM.

• Incubate the plates overnight at 37°C.

• The next day, count the colonies to assess the effect of GCV on the viable cell counts.

3. Turbidity Changes and Viable Cell Count Measurement After GCV Addition:

• To each of the 6 pre-cultured tubes, add 50 µL of GCV stock (1000 µM final concentration).

• Shake the cultures at 37°C and measure the turbidity (OD600) every 30 minutes to monitor the growth over time.

• At 3 hours, 6 hours, and 9 hours after GCV addition, take aliquots from each culture and dilute them 10^6-fold.

• Plate 100 µL of each diluted sample onto LB plates and incubate overnight at 37°C.

• After incubation, count the colonies to assess the viable cell count at each time point.

Data Analysis:

• Compare colony formation on GCV plates across different GCV concentrations (0 µM, 100 µM, and 1000 µM) between TK+ and TK- strains.

• Analyze the turbidity changes over time to determine the growth inhibition effect of GCV.

• Compare the viable cell counts at 3, 6, and 9 hours to evaluate the impact of GCV on cell survival in liquid culture.

Purpose:

The purpose of Assay 5 is to evaluate the immediate effects of Ganciclovir (GCV) on highly diluted TK+ and TK- strains. This assay focuses on observing the impact of GCV treatment on colony formation after short-term incubation.

Methods:

1. Dilution of Pre-cultured TK+ and TK- Strains:

• Pre-cultured TK+ and TK- strains were diluted to 10^-8 in LB medium to significantly reduce the bacterial concentration.

2. GCV Addition and Incubation:

• Add GCV to the diluted bacterial suspensions to reach a final concentration of 100 µM.

• Incubate the GCV-treated suspensions at room temperature for 5 minutes.

3. Plating:

• Prepare the following sample groups for plating:

[TK-, GCV+]: TK- strain treated with GCV.

[TK+, GCV+]: TK+ strain treated with GCV.

[TK+, GCV-]: TK+ strain without GCV treatment (control).

• Plate each of these sample groups onto LB agar plates.

• Incubate the plates at 37°C overnight.

Data Analysis:

• The next day, count the colonies to assess the effect of GCV on both the TK+ and TK- strains.

• Compare the colony counts between the TK+, GCV+ and TK+, GCV- samples to evaluate the impact of GCV on the TK+ strain.

• The TK-, GCV+ group will serve as a control to confirm the specificity of GCV's action on the TK+ strain.

Purpose:

Based on dry modeling, it was suggested that E. coli requires a significant amount of time to uptake Ganciclovir (GCV). In response, Assay 6 was designed to evaluate the impact of prolonged incubation with GCV before plating and colony counting.

Procedure:

1. Sample Preparation:

The following sample groups were prepared:

• TK+, GCV+: TK+ strain with GCV treatment.

• TK-, GCV+: TK- strain with GCV treatment.

• TK+, GCV-: TK+ strain without GCV (control).

2. GCV Incubation:

After adding GCV to the appropriate samples, all groups were incubated at 37°C for 24 hours.

3. Plating and Colony Counting:

After the 24-hour incubation, the samples were plated onto LB agar plates, and the plates were incubated overnight at 37°C. Colony counting was performed the next day to assess the effect of GCV on both the TK+ and TK- strains.

Results

Fig 27. HSV-TK/GCV Assay6 Extend Incubation to Assess GCV Uptake in E.coli

The results from Assay 6 revealed a fivefold difference in the number of viable cells. Additionally, as suggested by the DryLab, it was confirmed that more than 24 hours are required for GCV uptake. For future prospects, extending the incubation time and measuring the number of viable cells may lead to more favorable results