Experiments

For quantifying PCR or other DNA products, the Qubit 1x dsDNA HS kit (Invitrogen) was used. 2 µL of 10-fold diluted DNA was added to 198 µL working solution and 10 µL standards (1 and 2) were added to 190 µL working solution. The standards were measured on the Qubit machine after which the samples were measured. Because 10-fold diluted DNA was used, results were corrected to the original (undiluted) concentrations.

Golden gate assembly was done according to the overnight conventional modular cloning methodology of the Joint Universal Modular Plasmid (JUMP) system (Valenzuela-Ortega & French, 2021).

Reactions were prepared for digestion and incubated for 1 hour at 37 °C. After this, ligation was performed according to the two-step assembly ligation methodology of the Joint Universal Modular Plasmid (JUMP) system (Valenzuela-Ortega & French, 2021). For ligation, a 1:6 molar ratio of backbone:insert was maintained.

After Golden Gate assembly, DNA was cloned into E. coli. To do this, competent DH5α cells were thawed on ice for 10-15 minutes. 1 µL of plasmid DNA or full Golden Gate Assembly was added to 50 µL of competent cells, mixed gently, and incubated on ice for 30 minutes. After this, cells were heat-shocked for 45 seconds at 42°C in a water bath. Cells were placed back on ice for five minutes. 500 µL of sterile LB medium was added to the cells, after which they were left to recover at 37°C for one to two hours. After recovery, cells were plated on appropriate antibiotic LB plates and incubated overnight at 37°C (14-18 hours).

2x 250 mL water agar was melted by microwaving for 2-3 minutes. Antibiotics and/or other supplements were added to 2x 250 mL growth medium, which was added to the melted agar and mixed well. 16 mL of agar mixture was poured in Petri dishes and left at room temperature to cool.

To determine how many bacteria were in our medium per mL for different ODs, we made multiple serial dilutions. As starting ODs we used approximately 1.5, 1.0, 0.75, 0.5, 0.25, and 0.1. These were then diluted 10³ to 10⁹ times in steps of 10x. This meant that we ended up with 6 ODs, all with 7 dilutions for a total of 42 dilutions. We then plated 50 µL of these solutions on agar plates with kanamycin. After growing them over the weekend at room temperature, we counted the colonies and multiplied it by the relevant factor to get a number of bacteria per mL of the starting OD.

After overnight incubation of transformations, colonies were picked for miniprepping and DNA sequencing to confirm the DNA sequences. Miniprep tubes with 5 mL of LB medium (with appropriate antibiotics) were inoculated with one colony of transformed bacteria, picked with the tip of a pipette. Colonies were grown overnight at 37 °C in a shaker at 180-200 rpm. The next day, cultures were miniprepped according to the QIAprep Spin Miniprep Kit (Qiagen). 5 µL of each sequencing primer of interest was added to 5 µL DNA and sent for sequencing.

Clones could also be analyzed by running an analytical digest on gel. A restriction enzyme was selected that enables differentiation of fragment sizes, allowing confirmation of the insert size. In our case, NotI enzyme was used for all our restriction digestions. A master mix was prepared for the number of reactions. 15 µL of the master mix was added to 5 µL DNA and incubated at 37°C in a block heater for 20-60 minutes; for Fast Digest enzymes, 20 minutes sufficed. Digestion products were run on a 1% agarose gel.

A 1% agarose gel with 200 mL 1x TAE and 2 g agarose was prepared by adding the agarose to 100 mL 1x TAE and melting it in the microwave for approximately 2 minutes until the mixture was boiling. Then, the remaining 100 mL 1x TAE was added. 7 µL Midori Green DNA stain (NIPPON Genetics Europe) was added and the mixture was poured into the tray with a comb. After setting the gel, the samples were loaded. For (colony) PCR products, 4 µL of TriTrack dye (ThermoFisher Scientific) was added to 20 µL sample. For restriction digestion products (done with Green Fast Digest buffer), only the 20 µL sample was loaded. 7 µL of a 1 kb DNA ladder (ThermoFisher Scientific) was added and the gel was run for 30-40 minutes at 110-120 V.

When transformation gave many colonies with similar phenotypes, instead of miniprepping and running an analytical digest, colony PCR was chosen as an initial screening method to filter the candidates. A colony PCR master mix was prepared and each PCR tube was filled with 20 µL of the master mix. Sterile culture tubes with the appropriate growth medium were prepared. Colonies were picked and resuspended by pipetting up and down in the PCR mix, after which the same tip was used to inoculate a culture tube. The PCR reactions were run on a thermocycler for 30 cycles (approximately 1.5 hours). Products were analyzed on a 1% agarose TAE electrophoresis gel with Midori Green dye (NIPPON Genetics Europe). Colonies yielding the correct band were selected, and their corresponding cultures were grown for plasmid preparation.

To change overhangs of gene blocks for level 2 assembly, PCR was used. A master mix was prepared for the number of reactions. The necessary amount of master mix was added to 2 ng DNA in PCR tubes. The PCR tubes were placed in a thermocycler and the PCR program was run for 30 cycles. The PCR products were run on a 1% agarose TAE gel and DNA concentrations were determined using Qubit quantification (Invitrogen).

After validation of constructs in E. coli, they were also introduced into P. alcaligenes. P. alcaligenes cells were made electrocompetent using a microcentrifuge-based procedure. 50 mL of culture was spun down at room temperature for 20 min at 4000 rpm. Pellets were resuspended in 10 mL 300mM sucrose, then spun down again for 20 mins at 4000 rpm. The pellet was then resuspended in 2 mL of 300 mM sucrose. For electroporation, 5 µg of DNA was mixed with 100 µL of electrocompetent cells. The mixture was transferred to an electroporation cuvette and electroporated with settings: 25 μF; 200 Ω; 2.5 kV. After electroporation, cells were transferred to a microcentrifuge tube and 1 ml of room temperature LB medium (without antibiotics) was added. Cells were left to recover for 1.5 hours at 180-200 rpm at 37 °C. After recovery, cells were plated on appropriate antibiotic LB plates and incubated overnight at 37°C. For P. alcaligenes, growing of colonies could take up to 2 days.

Reference: Valenzuela-Ortega, M., & French, C. (2021). Joint universal modular plasmids (JUMP): A flexible vector platform for synthetic biology. Synthetic Biology, 6(1), ysab003.

In a reaction vessel with a total volume of 100 mL, bacterial culture broth, 5g of ammonium acetate, 0.2g of sodium sulfite, 0.5g (0.5%) of sodium pyruvate, 0.6 g of pyrocatechol and 0.1g of EDTA were combined. The pH of the culture was adjusted to 8 via ammonium acetate and kept constant throughout. The culture was incubated at 15 °C, kept away from light exposure. The culture was fed every two hours with 0.5g (0.5%) of sodium pyruvate and 0.6 g of pyrocatechol to maintain initial concentration. Production of L-DOPA could take between 48-72 hours.

In a reaction vessel with a total volume of 100 mL, bacterial culture broth, 5g of ammonium acetate, 0.2g of sodium sulfite, 0.5g (0.5%) of sodium pyruvate, 0.6 g of pyrocatechol and 0.1g of EDTA were combined. The pH of the culture was adjusted to 8 via ammonium acetate and kept constant throughout. The culture was incubated at 5 °C, covered in foil and in a closed styrofoam container. The culture was fed every two hours with 0.5g (0.5%) of sodium pyruvate and 0.6 g of pyrocatechol to maintain initial concentration, from 9:00-17:00. Production of L-DOPA was sustained for 3-4 days straight.

Safety: Handle cultures with care in a fume hood. PPE (gloves, lab coat, and goggles) should be worn at all times. For further detail, look up catechol safety guidelines.

4.0 mg of L-DOPA and 0.6 mg of methyl-DOPA were dissolved in 2 mL of MilliQ water (vortexed for 60 seconds, sonicated for 30 minutes). When no visible particles were visible, 8 mL of 100% HPLC grade acetonitrile (ACN) was added to each sample and mixed until well incorporated.

Samples were analyzed in the SHIMADZU HPLC system (Shimadzu SPD M40 diode array detector operated between 210-400nm), with the Waters HILIC BEH Amide 5µm, 4.6x100mm column. The detector operated at 280nm, with a flow rate of 0.8 mL/min, and the mobile phase 80% acetonitrile (HPLC grade) + 0.1% formic acid.

Once individual retention times were recorded, the samples were combined 50/50 to determine if there was enough separation between them to use methyl-DOPA as a future internal standard (IS).

The protocol we developed draws on ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, as adopted by the EMA, but does not constitute a complete ICH validation. It is worth noting that this protocol can be used for quantification of other polar molecules. If the sample of interest is below the limit of detection (LOD), this method can be used in LC-MS/MS with minor adjustments.

This is a valuable addition to the protocols established by the 2024 iGEM team CSMU-NCHU-Taiwan, which quantified and extracted L-DOPA from plants. Now, future iGEM teams have protocols for quantification of polar molecules in three different biological matrices: FBS, LB, and plant leaves (Nicotiana benthamiana).

HPLC Setup: SHIMADZU HPLC system (Shimadzu SPD M40 diode array detector operated between 210-400nm), with the Waters HILIC BEH Amide 5µm, 4.6x100mm column. The detector operated at 280nm, with a flow rate of 0.8 mL/min, stable at around 84 mbars. Mobile phase: 80% acetonitrile (HPLC grade) + 0.1% formic acid.

Prior to starting work, we purged the lines twice, purged the autosampler, and checked the retention times of L-DOPA, methyl-DOPA (internal standard), and methyl-DOPA + L-DOPA in 80% ACN. Then, we prepared separate samples of L-DOPA, methyl-DOPA, and L-DOPA + methyl-DOPA in LB. We used 100 µL of each sample, centrifuged for 10 minutes at 1200 Gs, and measured the supernatant.

Calibration Curve: The calibration curve was established based on ICH guideline M10 on bioanalytical method validation (04 April, 2024), which requires at least 6 dilutions with internal standard and two blanks - one with and one without internal standard. The same extraction method was used on all samples, except the blank without the internal standard.

Working Mix: 50 mL of LB, 2.5g ammonium acetate, 0.1g sodium sulfite, 0.05g EDTA, 0.3g catechol.

The protocol draws on ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, as adopted by the EMA, but does not constitute a complete ICH validation. Prior to quantifying L-DOPA in zebrafish larvae, we chose to quantify it in a replacement matrix to limit the use of zebrafish larvae.

This protocol outlines the first steps of method development for a complex biological matrix for HPLC. We have prepared some zebrafish larvae samples, but they are yet to be validated.

The protocol is virtually the same as for LB, with the same extraction method, calibration curve values, and HPLC setup. The HPLC setup: Detector operating at 280nm, flow rate 0.8 mL/min, column stable at around 84 mbars. Mobile phase 80% ACN + 0.1% FA. The L-DOPA, methyl-DOPA, and L-DOPA+methyl-DOPA standards in 80% ACN should be run first.

Much like in LB, first the protocol should be conducted on regular FBS and then FBS with working mix.

Working Mix (FBS): 50 mL of FBS solution, 2.5g ammonium acetate, 0.1g sodium sulfite, 0.05g EDTA, 0.3g catechol.

L-DOPA oxidation creates melanin and DOPAquinone, which are reddish-brown in color. To administer L-DOPA and carbidopa to the zebrafish larvae (<5dpf), both compounds were dissolved in E3 (embryo medium).

These concentrations were achieved by converting human working blood concentration for zebrafish larvae using the following calculation, considering that the minimal L-DOPA dosage in humans is 0.3g/day while the maximum is 1.2g/day:

Zebrafish mg/L = human g/day × 1000/6
Zebrafish mg/L = 50 - 200

However, in this instance we chose a concentration significantly higher, 4mg/mL of L-DOPA and 1mg/mL carbidopa - not a fatal concentration for the larvae but sufficient to produce a significant color change.

Serial Dilution: To assess the sensitivity of zebrafish larval color change to different L-DOPA concentrations, a serial dilution was prepared ranging from 1000 mg/L (with 250 mg/L carbidopa) down to 10 mg/L (with 2.5 mg/L carbidopa), plus three controls.

2 mL of each solution was administered into designated wells of 2 six-well plates, where each well contained 30 zebrafish larvae (<5 dpf). The larvae were then incubated in the dark at room temperature for 3 hours. Zebrafish larvae were then anesthetized, immobilized, and imaged using an inverted low-magnification tissue culture microscope. After the experiment, the larvae were euthanized as per laboratory and EU standards.

The method for quantification of L-DOPA in zebrafish larvae is not yet developed to the same level as the protocol for LB, due to the lack of time and resources. The calibration curve still needs to be made in a representative biological matrix.

This preliminary extraction approach provides a foundation for future development and optimization of L-DOPA quantification methods in zebrafish larvae samples.

A digital balance was used to measure out the chemicals needed to produce the working solution. The working solution was stored in a 15 mL tube covered in foil, since some compounds are photosensitive. If kept overnight, the solution was kept at 4°C. Separately, 3 mg of ascorbic acid was also measured out and kept in a foiled 50 mL tube at room temperature. Under sterile conditions in a cell culture hood, 45 mL of medium was prepared at room temperature. To the working solution, 5 mL of medium was added and vortexed. To the ascorbic acid, 34 mL of medium was added to achieve 500 µM concentration. Original medium was removed from the previously prepared cell cultures. The working solutions were added to the cell cultures and incubated at 37°C for 4.5 hours in a sterile incubator. After incubation, the barrier integrity was reassessed using transepithelial/transendothelial electrical resistance (TEER). The supernatant was collected from apical and basolateral sides of the transwell for further LDH assessment to determine cell viability.

A digital balance was used to measure out the chemicals needed to produce the working solution. The working solution was stored in a 15 mL tube covered in foil, since some compounds are photosensitive. If kept overnight, the solution was kept at 4°C. To the working solution, 5 mL of medium was added and vortexed. A 2-fold serial dilution was then prepared. In the first well, 200 μL of the working dilution was added. From this well, 100 μL was transferred to the next and mixed with 100 μL of fresh medium to achieve a two-fold dilution. This process was continued across the plate to complete the dilution series. A 1% Triton X-100 solution in Milli-Q water was prepared as a positive control for cell lysis, while medium alone served as the negative control. All wells were incubated for 1 hour at 37°C in a sterile incubator. After incubation, 100 μL from each well was collected and transferred into Eppendorf tubes. Samples were either immediately frozen or kept on ice until further use. Lactate dehydrogenase (LDH) release was then measured using an LDH assay.

As the creation of these specific transwells is both time-consuming and requires a high level of expertise, the transwells used in our experiments were prepared by our supervisor, Francesco Suriano. Four transwell inserts were cultured under sterile conditions following the ALI-VIP protocol described by Floor et al. (2025). Briefly, Caco-2 cells were seeded onto collagen-coated transwell membranes and grown to confluency. After three days, the apical medium was removed to establish an air-liquid interface (ALI), while the basolateral compartment was supplemented with vasoactive intestinal peptide (VIP) to stimulate mucus production. The cells were differentiated for 11-14 days, resulting in polarized epithelial layers covered with a robust mucus layer. These prepared transwells provided the experimental system for our bacterial colonization and L-DOPA/GFP secretion studies.

Pseudomonas alcaligenes (L-DOPA producing) cultures were grown over the weekend at 37°C. E. coli strains (L-DOPA producing, GFP producing, and control) were grown at room temperature under the same conditions. From each culture, 5 mL was transferred to a sterile 15 mL tube. Optical density at 600 nm (OD600) was measured and converted to CFU/mL. Cultures were then transported in a sealed parafilm-covered transport box to the laboratory where the infection experiments were performed.

The infection volume was calculated according to the formula: V = (Caco-2 Cell number × Unit of infection)/CFU. As the exact number of Caco-2 cells was difficult to determine due to the mucus layer, we followed the reference protocol by Floor et al., using a unit of infection of 20, similar to that used for Salmonella infections. Since the calculated infection volume exceeded the capacity of the transwells, bacterial suspensions were centrifuged and resuspended in 500 µL of fresh medium. From this, 100 µL was added to each transwell for infection.

Following infection, cells were fixed with Carnoy's fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) for 2 hours at room temperature. After fixation, cells were washed once with 100% ethanol and rehydrated with 80% ethanol for 15 minutes before further processing.

To begin, the medium in the apical compartment of the transwell was carefully aspirated and replaced with 150 µL of Carnoy's solution (60% ethanol, 30% chloroform, 10% glacial acetic acid). The cells were fixed in this solution for 2 hours at room temperature. After fixation, the fixative was removed and the inserts were washed thoroughly with 100% ethanol. This was followed by a 15-minute incubation in 150 µL of 80% ethanol on the apical side. The ethanol was then replaced with PBS (150 µL apical, 600 µL basal), and the inserts were stored at 4°C.

Once equilibrated, the membranes were washed briefly in PBS. To permeabilize and block, 150 µL of blocking buffer was added to the apical compartment for 1 hour at room temperature. Two formulations of blocking buffer were prepared: one without Triton (2% BSA in PBS) and one containing Triton (2% BSA in PBS with 0.03% Triton X-100).

Primary antibodies were then prepared in blocking buffer. For the L-DOPA-producing bacteria samples (Pseudomonas GB7+8 and E. coli DH5α GB7+8), MUC2 antibody was diluted 1:100 (4 µL antibody in 396 µL buffer). For the GFP-producing bacteria (E. coli DH5α GB8+5 and the E. coli DH5α control), MUC2 was diluted 1:100 (2 µL antibody in 198 µL buffer), followed by the addition of Tubulin antibody (2 µL) to the same solution. After removing the blocking buffer, 200 µL of the prepared primary antibody solutions were added to the apical side of the transwell inserts. The samples were incubated for 1 hour at room temperature, protected from light. Following incubation, inserts were washed three times in blocking buffer (200 µL apical, 1000 µL basal, 5 minutes each wash).

Secondary antibody and nuclear stain preparations were then made. For the L-DOPA-producing bacteria samples, DAPI was diluted 1:500 (2.5 µL antibody in 499 µL buffer). For the GFP-producing bacteria and controls, a mix of Tubulin antibody (1:100) and DAPI (1:500) was prepared in blocking buffer (494 µL buffer, 1 µL DAPI, 5 µL Tubulin). In both cases, 200 µL of the secondary solution was added to the apical side, and the inserts were incubated for 30 minutes at room temperature in the dark.

The membranes were washed once with MilliQ water before being prepared for mounting. Using tweezers, the transwell inserts were removed and inverted. A scalpel was used to carefully cut out the membranes, with adjustments in angle and cutting speed made to prevent curling. Each membrane was transferred onto a glass slide containing a droplet of mounting solution (cell side facing up). An additional droplet of mounting solution was applied to cover the membrane fully, after which a coverslip was gently placed on top. The prepared slides were allowed to cure overnight at room temperature in the dark, stored horizontally to ensure even mounting.

Imaging: Images were acquired using a spinning disk Olympus SpinSR10 system equipped with a Yokogawa W1-SoRa spinning disk mounted on an IX83 stand with a Hamamatsu ORCA-Fusion camera. All imaging was performed in confocal mode using a 40× oil immersion objective. Z-stacks were collected to capture the full depth of the cell layers. Fluorescence signals from DAPI, MUC2, and mCherry were recorded sequentially using all available lasers (405, 488, 561 and 640 nm) with appropriate emission filters and exposure settings. The primary dichroic mirror used was a quadband (D405/488/561/640 nm). Single-color images were imported into cellSens Dimension software and combined as composite images. Orthogonal views were generated within cellSens. Imaging was performed with support from the Cell Imaging Core (CCI) at Utrecht University.

Experiments were started after feeding cycles were completed. Mating aquariums were prepared consisting of a main tank, inset, window, and cover. Tanks were filled with water to the border of the main tank. Fish were removed from their system tank. Using a net, females were transferred to one side of the inset and males to the other, taking care not to startle the fish. Tanks were placed back into the system and left overnight until the (artificial) sunrise.

The next morning, a fresh tank (without 'window') was filled with system water supplemented with filtered rainwater (RO water). The inset containing the fish was transferred into this fresh tank. Fish were left for approximately 30 minutes to allow egg laying. Afterward, the inset with fish was transferred back to another tank of water, and the eggs were collected. Eggs were washed with RO water and transferred to a Petri dish containing 30 mL of warm (28°C) E3 medium.

Eggs were transferred from the Petri dish into a 50 mL tube. Excess liquid was removed, and the eggs were washed twice with 20 mL sterile E3. They were then resuspended in 10 ml of sterile E3 containing antibiotics (Ampicillin 100 µg/mL, gentamicin 10 µg/mL, chloramphenicol 1 µg/mL). Culture dishes were prepared with 20 mL of sterile E3 supplemented with antibiotics, and the eggs were transferred into these dishes. The eggs were incubated for at least 6 hours at 28°C.

Eggs were transferred from the culture dish into a 50 mL tube. A bleach dilution of 1:1600 (0.003%) was prepared in sterile E3 from a 5% sodium hypochlorite bleach stock. Eggs were washed with 20 mL sterile E3, followed by a wash with 20 mL bleach dilution for 2 minutes; this was repeated once. They were then washed twice more with 20 mL sterile E3. Eggs were transferred to new culture dishes containing sterile E3.

To release the embryos from the chorion, sharp tweezers were inserted into the egg, expanded slightly to puncture the chorion, and then withdrawn carefully. The freed larvae were transferred to new dishes with sterile E3.

50 mL of overnight bacterial culture was pelleted by centrifuging for 20 minutes at 4000 rpm. The pellet was washed with 2 mL of E3 and transferred to a microcentrifuge tube. In total, the pellet was washed twice with 2 mL of E3 by resuspending and centrifuging at 8000 rpm for 1 minute in a table top centrifuge. The final pellet was resuspended in 2 mL of E3, resulting in a concentrated bacterial culture.

Approximately 10 larvae were transferred to new plates containing 9.5 mL of sterile E3. To each plate, 500 µL of concentrated bacterial culture (2 × 10¹⁰ bacteria/mL) was added. Fish were inoculated until microscopy.

To anesthetize the larvae, tricaine was added to sterile E3 in a dish (final concentration of 1x Tricaine). Larvae were transferred into the dish, and once sufficiently sedated, were moved into imaging rings using a plastic pipette. Excess liquid was removed from the imaging ring. The cover slip was coated with 0.4% agarose containing tricaine, and the larvae were positioned at the center and gently pushed to the bottom against the slip.

Live imaging was performed on a Spinning Disc (Yokogawa CSU-X1-A1) Nikon Eclipse Ti microscope with Perfect Focus System equipped with a sample incubator (Tokai-Hit) and an Evolve 512 EMCCD camera (Photometrics), controlled with MetaMorph 7.7 software (Molecular Devices). Imaging was carried out with a 60× water-immersion objective (Plan Apo VC, NA 1.2; Nikon) and a 20× dry objective (Plan FL LWD, NA 0.70; Nikon), using Cobolt Calypso 491 nm and Cobolt 647 nm lasers and brightfield (light source) for excitation.

Larvae colonized with arabinose-induced kill switch-containing bacteria were imaged. First, a tile scan of the fish was taken, followed by an initial Z-stack to document stable gut colonization in the anterior-middle gut. To induce the kill switch, 250 µM L-arabinose was added on top of the agar slab until the solution reached the rim of the imaging ring. A coverslip was carefully placed on top and gently pressed until flat, ensuring a uniform layer of L-arabinose covered the agar slab; any excess solution was removed by pipetting. Overnight Z-stack imaging of gut colonization was then performed at the same locus as before, for a total of 16 hours, acquiring Z-stacks of the fluorescence channels every 15 minutes.