Team:ETH Zurich/Collaborations

We participated in several collaborations with teams from Europe and South America. The iGEM team from Marburg carried out an InterLab study with Vibrio natriegens to show the potential of this organism to increase the speed at which new findings in synthetic biology can be generated. Another collaboration we had was with the Unesp Brazil team. Thanks to our experience with modeling we were happy to help them improve on their own modelling part. Finally, we had a collaboration with the iGEM team from Utrecht. They engineered the Tar receptor to establish different sensing thresholds. The intellectual exchange helped us to improve on our own project.

Marburg InterLab Study

We not only participated in the official fifth International InterLaboratory Measurement Study in synthetic biology, which is carried out by the iGEM competition itself but also in the InterLab study from this year’s Marburg iGEM team. They are working with Vibrio natriegens as a possible new chassis for synthetic biology. In their project „Vibrigens - Accelerating Synbio“ they aim to establish Vibrio natriegens, the fastest-growing organism known to date (doubling time ~ 10 min) as the new go-to organism for everyday lab work in both academic and industrial applications. The task in their InterLab study was similar to the official iGEM InterLab study, i.e. to measure fluorescence of different test devices at different time points.

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Show Protocols

Workflow

Workflow of the Marburg InterLab study. Two colonies from each transformation are picked and a day culture is inoculated at 37°C. At two time-points (0h and 3h) a sample is taken and analysed in terms of OD₆₀₀ and fluorescence intensity

Cell measurement

Test Devices used for transformation and subsequent fluorescence measurements.

Device	Part number
Negative control	BBa_R0040
Positive control	BBa_I20270
Test Device 1	BBa_J364000
Test Device 2	BBa_J364001
Test Device 3	BBa_J364002 B
Test Device 4	Ba_J364007
Test Device 5	BBa_J364008
Test Device 6	BBa_J364009

Depicts the plate layout for the fluorescence and OD₆₀₀ measurements. For both time-points (0h and 3h) a plate is measured.

Fluorescence at 0h

	Negative Control	Positive Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.0120	0.0153	0.0134	0.0153	0.0182	0.0221	0.0196	0.0172
Colony 1, Replicate 2	0.0127	0.0157	0.0132	0.0150	0.0164	0.0218	0.0123	0.0197
Colony 1, Replicate 3	0.0135	0.0167	0.0135	0.0157	0.0139	0.0216	0.0120	0.0129
Colony 1, Replicate 4	0.0153	0.0174	0.0139	0.0146	0.0243	0.0221	0.0121	0.0145
Colony 2, Replicate 1	NA	0.0251	0.0184	0.0143	0.0147	0.0143	0.0122	0.0143
Colony 2, Replicate 2	NA	0.0286	0.0145	0.0142	0.0148	0.0149	0.0210	0.0142
Colony 2, Replicate 3	NA	0.0273	0.0165	0.0146	0.0151	0.0145	0.0163	0.0147
Colony 2, Replicate 4	NA	0.0316	0.0152	0.0154	0.0155	0.0175	0.0204	0.0153

Fluorescence at 0h

	Negative Control	Positive Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	291.4	401.4	294.4	173.4	128.4	316.4	239.4	116.4
Colony 1, Replicate 2	209.4	273.4	170.4	121.4	37.4	290.4	177.4	55.4
Colony 1, Replicate 3	218.4	285.4	200.4	122.4	70.4	270.4	217.4	101.4
Colony 1, Replicate 4	197.4	306.4	202.4	142.4	72.4	305.4	182.4	88.4
Colony 2, Replicate 1	NA	255.4	154.4	165.4	155.4	113.4	181.4	87.4
Colony 2, Replicate 2	NA	252.4	146.4	211.4	113.4	123.4	187.4	90.4
Colony 2, Replicate 3	NA	296.4	156.4	285.4	200.4	157.4	244.4	101.4
Colony 2, Replicate 4	NA	353.4	267.4	262.4	240.4	201.4	292.4	141.4

OD₆₀₀ at 3h

	Negative Control	Positive Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.455	0.449	0.374	0.486	0.487	0.416	0.473	0.391
Colony 1, Replicate 2	0.448	0.445	0.368	0.483	0.478	0.410	0.460	0.384
Colony 1, Replicate 3	0.451	0.432	0.357	0.472	0.470	0.400	0.456	0.379
Colony 1, Replicate 4	0.442	0.429	0.356	0.469	0.468	0.397	0.455	0.375
Colony 2, Replicate 1	NA	0.607	0.327	0.362	0.457	0.403	0.391	0.496
Colony 2, Replicate 2	NA	0.604	0.326	0.357	0.451	0.398	0.390	0.492
Colony 2, Replicate 3	NA	0.601	0.325	0.356	0.449	0.394	0.388	0.490
Colony 2, Replicate 4	NA	0.606	0.327	0.355	0.455	0.396	0.391	0.492

Fluorescence at 3h

	Negative Control	Positive Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	1153.125	10515.125	9225.125	3968.125	1367.125	11505.125	27585.125	1512.125
Colony 1, Replicate 2	1167.125	10514.125	8943.125	3864.125	1297.125	11149.125	26946.125	1438.125
Colony 1, Replicate 3	1199.125	10421.125	9024.125	3917.125	1311.125	11161.125	27287.125	1420.125
Colony 1, Replicate 4	1155.125	10507.125	8991.125	3856.125	1295.125	11337.125	27715.125	1424.125
Colony 2, Replicate 1	NA	9132.125	9758.125	2786.125	1261.125	5555.125	28023.125	2015.125
Colony 2, Replicate 2	NA	9162.125	9925.125	2769.125	1232.125	5543.125	27866.125	1974.125
Colony 2, Replicate 3	NA	9162.125	10162.125	2875.125	1264.125	5609.125	28439.125	2046.125
Colony 2, Replicate 4	NA	9450.125	10366.125	2937.125	1326.125	5779.125	29401.125	2088.125

Protocols

Preparation of electrocompetent cells

Introduction

This protocol was provided by the Marburg iGEM team to perform their InterLab study.

Materials

LB media + v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2)
Electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH = 7) (sterile filtrated)
Overnight culture of Vibrio natriegens inoculated from plates or cryostock in LB + v2 salts (e.g. 5mL, 37 °C; at 200 r.p.m) ( 10mL Falcon from the box )
Pre-chill the electroporation buffer (approx. 500 mL) and the centrifugation tubes you want to use (on ice)
On the following day, 500 mL of the LB + v2 is inoculated with the overnight culture with a final OD of 0.05
The culture is grown at 37 °C in a baffled flask with shaking at 200 r.p.m. until an OD600 of 0.5

Procedure

Divide the culture into two (or more) pre-chilled centrifugation containments
Put the culture on ice for 15 min
The cells are pelleted at 3000x g. for 20 min at 4 °C to avoid cell damage
The supernatant is carefully decanted and the cell pellets of both containers are gently resuspended in 10 mL of chilled electroporation buffer
The suspensions are transferred to two chilled 50 mL Falcons
The tubes are each filled to top with additional chilled electroporation buffer
(max. 50 mL in total or fill to maximum volume in accordance with manufactures instruction)
The Falcon tubes are inverted several times
The cells are centrifuged down at 3000x g for 15 min at 4 °C
The washing step (resuspend, fill-up, invert, centrifuge, discard supernatant) is repeated two times for a total of three washes• After the final wash, the supernatant is carefully decanted and the cells are gently resuspended in residual electroporation buffer (equals approximately 500 µL)
Measure the OD in a 1:20 dilution against electroporation buffer (e.g. 950 µL buffer & 50 µL sample)
The volume is adjusted with additional electroporation buffer to bring the final OD600 to 16
Cells are aliquoted (80 µL) into chilled tubes, frozen in liquid Nitrogen and stored at −80 °C until use

Evaluation

Competent cells were used for electroporation at 0.9 kV to successfully transform cells.

Results

20 aliquots were stored at -80°C in the freezer (room 3.80)

Electroporation

Introduction

This protocol was provided by the Marburg iGEM team to perform their InterLab study with Vibrigens.

Materials

Recovery media 10 aliquots (a’ 500µL )(BHI + v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2), and 680 mM sucrose)
Preheat the aliquots with the recovery medium to 45 °C ( pipetting mixing with cold cells and resuspending in chilled cuvettes will cool down the media )
Agar plates (LB + v2 + 1% agarose + 2 µg/ml chloramphenicol (Cm))

Procedure

Take nine aliquots of the prepared competent cells and place them on ice.
1µL of the Plasmid DNA (100 ng/µL) and the electrocompetent cells are combined and gently mixed (NO VORTEX, avoid foaming) in a chilled 1.5 mL microcentrifuge tube.
(each aliquot gets one 1 µL of another plasmid from the box one aliquot is a control and will later not be plated out on a plate with antibiotics)
The cell-DNA suspension is transferred to a chilled electroporation cuvette with a 0.1 cm gap size
Cells are electroporated with the following settings: 900 V, 25 mF, 200 Ω (Eppendorf electroporator)
Cells are immediately recovered in 500 μL (one aliquot) preheated (45°C) recovery medium and transferred to a 1.5 mL tube
The cells are recovered by incubating at 37 °C for 1.5 h. (also put the agar plates for preheating in the incubator at 37 °C)
The cells are centrifuged down at 3000x g, most of the supernatant is removed
The pellet is resuspended in the leftover media (approx. 50 µL) and plated out on warm agar plates (37 °C) containing the appropriate antibiotic (2 µg/ml Chloramphenicol - Cm)
The plates are incubated for several hours or overnight at 37 °C for colonies to appear. Check the rest of the InterLab Protocol to keep track of colony growth

Evaluation

Electroporation with all test devices worked and yielded colonies

Results

Transformation with test devices was successful.

Cell measurement

Introduction

This protocol was provided by the Marburg iGEM team to perform their InterLab study with Vibrigens. Deviations from the original protocol are highlighted.

Materials

LB media + v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2)
Plate reader

Procedure

Pick two colonies from each of the transformation plates and inoculate in 5 mL LB + v2 + 2 µg/mL Chloramphenicol. Grow the cells overnight (16-18 hours) at 37 °C and 220 r.p.m. 10mL cultures were first grown 24h at RT and subsequently for another 1h at 37°C
Make a 1:20 dilution of each overnight culture in 0.25 mL of culture into 4.75 mL of LB + v2 + 2 μg/mL Chloramphenicol
Measure Abs600 of 200 µL of these diluted cultures and with a blank in a 96-well plate (samples should be laid out according to the plate diagram below)
Record the data in your notebook
Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 mL LB + v2 + 2μg/mL Chloramphenicol in 50 mL Falcon tube (amber, or covered with foil to block light)
Take 500 μL samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 3 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice
Incubate the remainder of the cultures at 37 °C and 220 r.p.m for 3 hours
Take 500 μL samples of the cultures at 3 hours of incubation into 1.5 ml Eppendorf tubes. Place the samples on ice
At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement). Exitation wavelength: 485nm (Bandwidth 20nm), Emission wavelength: 535nm Bandwidth(20nm).
Record data in your notebook

Evaluation

Colony 2 of the negative device did not grow, therefore no data is available for these replicates! Further evaluation is performed by the Marburg InterLab team. The data was sent to the Marburg iGEM team is presented in the tables 2-4.

Results

Results are published by the Marburg iGEM team. Please refere to their webpage.

Unesp Brazil

The iGEM team Unesp Brazil works on a new framework to engineer and test probiotics. To analyze their circuit, they use modelling. A major concern was the production of insulin in the absence of glucose. We helped them to improve their model and supported them throughout the develpment of their model. Our contribution in particular:

We helped to improve their ODE model by adding additional equations and restrictions to their model. Compare Figure 1 and Figure 2 to see how this influenced their model output. As you can see we were able to adjust their model so that it predicts reasonable concentrations. In Figure 1.2 the K_D for sRNA/DNA binding is depcited and shows how important it is to optimize this paramter for their model in order to obtain the desired output.
We developed guidlines for the team to guide thier parameter estimation and helped them with the literature research for the parametres of their models.
The Unesp Brazil team is working on a booklet on modelling for further iGEM teams at their university. This booklet is meant to guide subsequent teams on their modelling approaches. We were happy to contribute by providing the Tips & Tricks section in this booklet.
We provided lecture material for stochastic modelling.

Show Modelling Graphs

On-state of the improved model.

Off-state of the improved model.

Model output after optimization.

On-state of the initial model.

Off-state of the initial model.

Initial model output

Utrecht

This year, the iGEM team from Utrecht is also working on modifying and improving the Tar receptor of E. coli. In their project they are evolving the natural receptor so that it can reliably detect pollutants in water. Furthermore, they are planning to modify the receptor to increase the range of detectable molecules, making it more applicable in real life. During the European iGEM Meetup in July, we realized that both of our teams were working on chemotaxis-based biosensors. Although our projects are based on somewhat different methods and applications, we did struggle with similar complications at the time. Therefore, we decided to stay in touch and update each other about problems and solutions, which formed the base for our collaboration. An example of our similar approaches was found in the way both our teams dealt with our intended use of split luciferase. Both our projects included using the reconstitution of a split luciferase to create a read-out signal from the chemotaxis pathway. Especially when searching for the right luciferase for our application, we were happy to split tasks. We shared outcomes with each other, which led to our current solution using firefly luciferase. In the future, we also aim to integrate the engineered bacteria from the Utrecht iGEM team on AROMA. We hope their biosensor will improve the range of detectable concentrations, while also broadening the range of substrates that could potentially be sensed.