Team:Toulouse INSA-UPS/Methods

Medium

LB∕LBA

Materials

10 g/L tryptone
5 g/L yeast extract
5 g/L NaCl
(15 g/L agar bacteriological agar type E)

Dissolve in demineralized water, stir and autoclave.

YPD/YPDA

Materials

10 g/L yeast extract
20 g/L bacto-peptone
20 g/L glucose or galactose
(20 g/L bacteriological agar type E)

Dissolve in demineralized water, stir and autoclave.

BGM (from the Marburg 2019 team)

Materials

Stock 1 (100X): (autoclave)

Material	Concentration
Na₂Mg EDTA	4.84 g/L
Ferric ammonium citrate	6.29 g/L
CaCl₂ x 2 H₂O	17.94 g/L
Citric acid x 1 H₂O	3.00 g/L

Stock 2 (100X): (autoclave)

Material	Concentration
MgSO₄ x 7 H₂O	37.46 g/L

Stock 3 (100X): (autoclave)

Material	Concentration
K₂HPO₄	10.45 g/L
KH₂PO₄	8.17 g/L

(Stock 4 (500X): (autoclave)

Material	Concentration
H₃BO₃	7.14 g/L
MnCl₂ x 4 H₂O	4.52 g/L
ZnSO₄ x 7 H₂O	311 mg/L
CuSO₄ x 5 H₂O	126 mg/L
NaMoO₄	122 mg/L
CoCl₂x 6 H₂O solve first in 30 mL water and add to stock 4	975 mg/L

Combine stock solutions and add 1 M HEPES/NaOH buffer (pH 8.0) stock solution to a final concentration of 20 mM. Add 8.16 g of NaNO₃ per liter of medium.
For solid media add 1.4 % Agar.
Autoclave. Add filter sterilized Na₂S₂O₃ to a final concentration of 1 mM to 5X BGM-Agar.

Co-culture medium

Our co-culture medium was made with BGM 1X medium to which we added:

Material	Concentration
Yeast extract w/o amino acids	1.2 g/L
NaCl	100 nM

Tips

We could have filtered the solutions

BG−11

In order to realize our BG11 we ordered the medium in powder form from PhytoTech Lab.
The medium was then prepared by mixing 1.67 g of powder in 1L of H₂O milliQ The solution was then autoclaved before use.

Electrophoresis gel (0.8%):

Dissolve 8 g/L agarose D-5 in TAE 1X

Molecular biology

GeneJET Plasmid Miniprep Kit (ThermoFischer)

Materials

1.5 mL Eppendorf tubes
Table-top Centrifuge
Kit Resuspension Solution
Kit Lysis Solution
Kit Neutralization Solution
Kit Wash Solution
Kit Elution Buffer

Protocol

Resuspend cells, lyse and neutralize:
Add to the pelleted cells:
250 μL of Resuspension Solution and vortex
250 μL of Lysis Solution and invert the tube 4-6 times
350 μL of Neutralization Solution and invert the tube 4-6 times
Centrifuge 5 minutes at 12,000 g
Bind DNA:
Transfer the supernatant to the Thermo Scientific GeneJET Spin Column.
Centrifuge 1 minute at 12,000 g
Wash the column:
Add 500 μL of Wash Solution and centrifuge for 30-60 s at 12,000 g
Discard the flow-through
Repeat twice
Centrifuge empty column for 1 minute at 12,000 g to get rid of Wash buffer
Elute purified DNA:
Transfer the column into a new tube
Add 50 μL of Elution Buffer to the column and incubate 2 minutes
Centrifuge 2 minutes at 12,000 g
Collect the flow-through

Tips

Wait ~10 min with the tube open to remove all the remaining ethanol

Quantity of DNA recovered (with a Nanodrop)

PCR

Materials

PCR thermocycler
0.2 mL PCR tubes
Primers (both forward and feverse)
Template DNA
DNA polymerase
Nuclease-Free Water

Using ™ NebCalculator is highly recommended.

Q5

This polymerase was used to amplify our fragments in order to perform our In Fusion.

Component	25 μL	50 μL	Final concentration
Q5 High-Fidelity 2X Master Mix	12.5 μL	25 μL	1X
10 μM Forward primer	1.25 μL	2.5 μL	0.5 μM
10 μM Reverse primer	1.25 μL	2.5 μL	0.5 μM
Template DNA	variable	variable	0.5 ng genomic 100 pg vector
Nuclease-Free Water	QSP 25 μL	QSP 50 μL

OneTaq

This polymerase was used for screening to verify the sizes of our fragments.

Step	Temperature	Time
Initial denaturation	98°C	30 seconds
25-35 Cycles
	98°C	10 seconds
	50-72°C	30 seconds
	72°C	30 seconds/kb
Final extension	72°C	2 minutes
Hold	4-10°C

CloneAmp

This polymerase was used for screening to verify the sizes of our fragments.

Component	25 μL	50 μL	Final concentration
One Taq Quick-Load 2X Master Mix	12.5 μL	25 μL	1X
10 μM Forward primer	0.5 μL	1 μL	0.2 μM
10 μM Reverse primer	0.5 μL	1 μL	0.2 μM
Template DNA	variable	variable	<1,000 ng
Nuclease-Free Water	QSP 25 μL	QSP 50 μL

Step	Temperature	Time
Initial denaturation	94°C	30 seconds
25-35 Cycles
	94°C	30 seconds
	45-68°C	45 seconds
	68°C	1 minute/kb
Final extension	68°C	5 minutes
Hold	4-10°C

Bacteria colony PCR

To perform colony PCR, colonies of bacteria were collected, thanks to toothpicks, on boxes after 1-2 days of incubation. The toothpicks were then directly plunged into the PCR tubes with the PCR mix. The tubes were then put in the thermocyclers to perform the PCR.

Gel verification and EtBr staining procedure

Agarose gel migration at 100V during 25 min using Gel Loading Dye, Purple (6X) by NEB, and 1kb plus ladder as molecular weight marker by NEB.
After migration of PCR products on 0.8% agarose gels, the gels were treated in BET baths for 10 minutes to stain the DNA and to reveal them under UV light.

NucleoSpin-Gel-and-PCR-Clean-up kit by NucleoSpin

Materials

Kit buffers NT1 and NT3
H₂O milliQ
1.5 mL Eppendorf tubes
Table-top centrifuge

Protocol

Excise DNA fragment and solubilize gel slice in twice the weighted volume with NT1
Incubate 10 min at 50°C
Centrifuge 30 s at 11,000 g
Add 700 μL of NT3, and centrifuge 30 s at 11,000 g
Repeat step 4
Centrifuge 1 min at 11,000 g
Add 20 μL of milliQ water, wait for 2 min at room temperature
Place the column into a new 1.5 mL eppendorf and centrifuge 1 min at 11,000 g

In Fusion HD Cloning Kit (Takara)

Materials

Stellar competent cells
Falcon tube
42°C water bath
Kit SOC medium
37°C incubator with and without agitation
Thermcycler
Kit control insert
Kit control vector
5X In-Fusion HD Enzyme Premix

Protocol

Purify fragment (insert or linearized vector) by gel extraction followed by spin-column purification using a silica-based purification system, such as the NucleoSpin Gel and PCR Clean-Up kit

Plan the In-Fusion cloning reaction. Good cloning efficiency is achieved when using 50–200 ng of vector and inserts, respectively. More is not better. Use the table below for reaction recommendations

Component	Cloning	Negative Control	Positive Control
Purified PCR fragment	10-200 ng		2 μL of 2 kb control insert
Linearized vector	50-500 ng	1 μL	1 μL of pUC19 control vector
5X In-Fusion HD Enzyme Premix	2 μL	2 μL	2 μL
Deionized Water	QSP 10 μL	QSP 10 μL	QSP 10 μL

Set up the In-Fusion cloning reaction:
2 μL 5X In-Fusion HD Enzyme Premix
x μL* Linearized vector
x μL* Purified PCR insert
x μL* Purified PCR insert
x μL dH₂O (as needed)
10 μL Total volume
* For reactions with larger combined volumes of vector and PCR insert (>7 μL of vector + insert), double the amount of enzyme premix, and add deionized water H₂O to a total volume of 20 μL
Incubate the reaction for 15 min at 50°C, then place on ice

Tips

This time can be increased to 30 minutes
Thaw Stellar Competent Cells in an ice bath just before use
After thawing, mix gently to ensure even distribution, and then transfer 50 μL of competent cells into a 14-mL round-bottom tube (falcon tube). Do not vortex
Add no more than 5 ng of DNA for transformation.
Place the column into a new 1.5 mL eppendorf and centrifuge 1 min at 11,000 g
Place tubes on ice for 30 min
Heat shock the cells for exactly 45 s at 42°C and then on ice for 1-2 min
Add SOC medium to bring the final volume to 500 μL. SOC medium should be warmed to 37°C before using
Incubate by shaking (160–225 rpm) for 1 hr at 37°C
Plate an appropriate amount of culture on a selective medium
NOTE: For a plate with a diameter of 9 cm, plate 100 μL. Plating is accomplished by spreading cells on selective medium [e.g., LB agar + Ampicillin (50–100 μg/mL)]. The medium should also contain X-Gal (40 μg/mL) for plasmids that permit blue/white screening of transformants
Incubate overnight at 37°C

Enzymatic digestion

Materials

NEB restriction enzymes
Template DNA
NEB Cutsmart Buffer 10X
37°C incubator

Protocol

Cutsmart Buffer 10X 2 μL
DNA around 100 ng
Enzyme 1 μL
H₂O milliQ qsp 20 μL
2h incubation at 37°C

Mix2Seq Kit NightXpress (eurofins Genomics)

Materials

Eurofins barcoded tubes
Primer
Template DNA
H₂O milliQ

Protocol

In the barcoded tube, add 15 μL of purified DNA (the quantity of DNA should not exceed 100 ng, we recommend 75 ng).
Add 2 μL of primer (one tube for one primer) at a concentration of 10 ng/μL. The total volume should be 17 μL.
Once the results are received, align with the “Template alignment” on benchling in order to compare with reference sequence

Yeast protocol

Yeast integration protocol provided by the LRSV

Materials

100°C dry or water bath
Filter-sterile plate buffer (40% PEG4000, 100 mM LiAc, 10 mM Tris-HCl, 1 mM EDTA)
Filter-sterile 1 M DTT
YPD broth and YPD agar (with and without the selection parameter)
Denatured single stranded DNA (salmon sperm DNA) b(y heating 5 minutes at 100°C, then keep on ice before use)

Protocol

Grow a 5 mL overnight recipient yeast culture in a shaking heat chamber at 30°C (with or without antibiotic, depending on the yeast strain)

With the overnight culture (at OD=0.15), inoculate 100 mL of prewarmed YPD broth. Grow to reach OD 0,6-1
Split the culture according to the number of constructs to be co-transformed in 50 mL falcon tube (one falcon = one transformation). Centrifuge 6 min at 6000 rpm
Remove the supernatant and add to the cells:
10 μL of SSDNA at a concentration of 10 mg/mL
1 μg to 2 μg DNA (optimum is 1 μg)
500 μL plate buffer
Transfer the mix in a 1.5 mL eppendorf and vortex vigorously
Add 20 μL of sterile DTT 1 M and vorte
Incubate at 21°C, 400 rpm overnight, as long as possible (15-20 hours)
Centrifuge 30 s at 5000 rpm
Remove the supernatant and gently resuspend the cells in 200-500 μL YPD
Spread on selective medium and incubate at 30°C for 2 to 5 days

Yeast Integration Takara Protocol

Materials

Yeast cells
YPD medium
Flask 1 L
30°C incubator with agitation
Table-top Centrifuge
1X TE/1X LiAc solution
Template DNA
Vortex
PEG/LiAc solution
DMSO
1X PEG solution
42°C water bath
Selective YPD agar plate

Protocol

Inoculate 1 mL of YPD with several 2–3 mm diameter colonies
Vortex vigorously for 5 min to disperse clumps
Transfer into a flask containing 50 mL of YPD or the appropriate SD medium
Incubate at 30°C for 16–18 hr with shaking at 250 rpm to reach stationary phase (OD600>1.5)
Transfer 30 mL of overnight culture to a flask containing 300 mL of YPD. Check the OD600 of the diluted culture and, if necessary, add more of the overnight culture to bring the OD600 up to 0.2–0.3
Incubate at 30°C for 3 hr with shaking (230 rpm). At this point, the OD600 should be 0.4–0.6
Place cells in 50-mL tubes and centrifuge at 1,000 g for 5 min at room temperature (20–21°C)
Discard the supernatant and thoroughly resuspend the cell pellets in sterile TE or distilled H₂O. Pool the cells into one tube (final volume 25–50 mL)
Centrifuge at 1,000 g for 5 min at room temperature.
Decant the supernatant.
Resuspend the cell pellet in 1.5 mL of freshly prepared, sterile 1X TE/1X LiAc
Add 0.1 μg of plasmid DNA and 0.1 mg of carrier DNA to a fresh 1.5-mL tube and mix.
Notes:
• For simultaneous cotransformation (using two different plasmids),use 0.1 μg of each plasmid (an approximately equal molar ratio),in addition to the 0.1 mg of carrier DNA.
• For transformations to integrate a reporter vector, use at least 1 μg of linearized plasmid DNA in addition to the carrier DNA.
Mix 0.1 mL of yeast competent cells to each DNA tube and mix well by vortexing
Add 0.6 mL of sterile PEG/LiAc solution to each tube and vortex at high speed for 10 s to mix
Incubate at 30°C for 30 min with shaking at 200 rpm
Add 70 μL of DMSO, mix well by gentle inversion (do not vortex)
Heat shock for 15 min in a 42°C water bath
Chill cells on ice for 1–2 min
Centrifuge cells for 5 s at 14,000 rpm at room temperature. Remove the supernatant
Resuspend cells in 0.5 mL of sterile 1X TE buffer
Plate 100 μL on each YPD agar plate that will select for the desired transformants. To ensure that you will obtain a plate with well-separated colonies, also spread 100 μL of a 1:1000, 1:100, and 1:10 dilution on YPD agar plates. These will also serve as controls for cotransformation efficiency. Note: If you are performing a cotransformation, plate controls to check transformation efficiency and markers of each plasmid. On separate 100-mm plates, spread 1 μL (diluted in 100 μL H₂O) on medium that will select for a single type of plasmid.
Incubate plates, upside-down, at 30°C until colonies appear (generally, 2–4 days).

Yeast colony PCR

Materials

Oese or toothpick
NaOH 20 mM
Thermocycler
Vortex
Table-top centrifuge

Protocol

Check the transformation plates after 2-5 days of growth at 30°.
Take a small amount of fresh biomass and resuspend in 15 μL of 20 mM NaOH
Incubate 15 min at 96°C
Vortex briefly and spin down the cells 5 minutes at 6000 g
Mix the following in a PCR tube (for 10 reactions):
50 μL 2xOneTaq Master Mix (New England Biolabs)
10 μM primer 1
10 μM primer 2
20 μL water
Add 1 μL of the denatured biomass to each tube containing 9 μL of the PCR premix
Run the following PCR program: 94°C for 1 min 35 cycles of [94°C for 20 seconds 50°C for 30 seconds 68°C for 1 min/kb of the PCR product] 68°C for 7 min 10°C hold
Analyze the products on 0.8% agarose gel

Cyanobacteria protocol

Transformation of the Helper Strain

Materials

Competent cells
1.5 mL Eppendorf tubes
Ice
42°C water bath
SOC medium
37°C incubator with and without agitation
Selective agar plates

Protocol

Thaw frozen competent cells on ice
Add 1-50 ng of DNA or 10 pg of competents cells control DNA (supplied as 0.1 ng/μL) per 50 μL tube of competent cells, quickly flick the tube several times (use the competent cells control DNA to determine transformation efficiency)
Keep tubes on ice for 30 minutes
Heat-shock the cells for 15-20 seconds in a water bath at exactly 42°C, do not shake
Immediately place the tubes on ice for 2 minutes
Add 450 μL of room temperature SOC medium to each transformation reaction and incubate for 60 minutes at 37°C with shaking.
For each transformation reaction, dilute the cells 1:10 and 1:100 and spread dilutions onto agar plates containing antibiotics. Incubate the plates at 37°C for 12-14 hours or overnight. If using BL21 (DE3) pLysS competent cells, do not dilute; spread 100 μL of these cells directly onto agar plates containing antibiotics.

Triparental conjugation protocol

Materials

Strains UTEX 2973, prK2013 and HB101
Table-top Centrifuge
37°C Incubator
40°C Incubator with high lighting
Sterile filters
BG11 agar plate with and without spectinomycin
BG11 broth
Flask 100 mL
Kanamycine
Sterile loop

Protocol

1. Growth of the cyanobacterial culture (day 1)

Prepare BG11 medium according to Lea-Smith et al., and agar plates with LB-BG11 and BG11+Kan50.
Set up a fresh culture of Synechocystis PCC 6803 or S. elongatus UTEX 2973 by inoculating a 100 mL conical flask of fresh BG11 medium (50 mL) with cells sourced from an axenic BG11 agar plate. Grow S. elongatus UTEX 2973 at 40°C, 300 μmol photons m^-2 s^-1 at 100 rpm. Grow cultures until OD_750nm = 0.5−1.5 (typically 1−2 days).

NOTE: S. elongatus UTEX 2973 cultures can be grown at 40°C in high light intensities (e.g., 2000 μmol photons m^-2 s^-1)

**2. Growth of helper and cargo E. coli strains (day 2)**

Inoculate LB medium containing ampicillin (final concentration 100 μg/mL) and chloramphenicol (final concentration 25 μg/mL) with a MC1061 E. coli strain containing vectors pRK24 and pRL528 (i.e., the helper strain) and grow at 37°C overnight at 225 rpm in a shaking incubator. Grow up a sufficient volume of helper strain culture, assuming 1 mL of culture is required per conjugation.
Inoculate LB medium (5 mL) containing appropriate antibiotics with the E. coli culture carrying the cargo vector (i.e., a Level T vector). Grow the culture at 37°C overnight at 225 rpm in a shaking incubator.

3. Conjugal transfer (triparental mating) (day 3)

Prepare the E. coli helper and cargo strains. Centrifuge the helper and the cargo E. coli overnight cultures at 3,000 g for 10 min at room temperature. Discard the supernatant without disturbing the cell pellet.
Wash the pellet by adding fresh LB medium without antibiotics. Use the same volume as the initial culture. Resuspend the pellet by gently pipetting up and down. Do not vortex the culture. Repeat this step 3x to remove residual antibiotics from the overnight culture.
Centrifuge the resuspended culture (as in step 3.3.1), discard the supernatant and resuspend in half the volume of LB medium of the initial culture volume (e.g., 2.5 mL if the overnight culture was 5 mL). Combine 450 μL of the helper strain with 450 μL of the cargo strain in a 2 mL tube and set aside (leave at RT) until step 3.3.6.
Prepare the cyanobacterial culture. For each conjugation reaction, use 1 mL of cyanobacterial culture (OD_750nm = 0.5−1.5).
Centrifuge the required total volume of cyanobacterial culture at 1,500 g for 10 min at RT, then discard the supernatant carefully without disturbing the cell pellet. Wash the pellet by adding fresh BG11 medium of the same initial volume. Resuspend the pellet by gently pipetting up and down, do not vortex the culture. Repeat this step 3x and set the washed culture aside.
Add an aliquot of washed cyanobacterial culture (900 μL) to the combined E. coli strains (helper and cargo) (900 μL) in a 2 mL tube. Mix the cultures by gently pipetting up and down. Do not vortex. Incubate the mixture at RT for 30 min for Synechocystis PCC 6803 or 2 hr for S. elongatus UTEX 2973.
Centrifuge the mixture at 1,500 g for 10 min at RT. Remove 1.6 mL of the supernatant. Resuspend the pellet in the remaining ~200 μL of supernatant. Place one 0.45 μm membrane filter on an LB-BG11 agar plate lacking antibiotics (section 8). Carefully spread 200 μL of the E. coli cyanobacterial culture mix on the membrane with a sterile spreader or a sterile bended tip and seal the plate with paraffin film
Incubate the LB-BG11 plate with the membrane for 24 hr. Maintain membranes with Synechocystis PCC 6803 cultures at 30°C, 100 μmol photons m^-2 s^-1. Maintain membranes with S. elongatus UTEX 2973 cultures at 40°C in 150 μmol photons m^-2 s^-1.

**2. Growth of helper and cargo E. coli strains (day 2)**

Inoculate LB medium containing ampicillin (final concentration 100 μg/mL) and chloramphenicol (final concentration 25 μg/mL) with a MC1061 E. coli strain containing vectors pRK24 and pRL528 (i.e., the helper strain) and grow at 37°C overnight at 225 rpm in a shaking incubator. Grow up a sufficient volume of helper strain culture, assuming 1 mL of culture is required per conjugation.
Inoculate LB medium (5 mL) containing appropriate antibiotics with the E. coli culture carrying the cargo vector (i.e., a Level T vector). Grow the culture at 37°C overnight at 225 rpm in a shaking incubator.

4. Membrane transfer

After 24 hr, carefully transfer the membrane using flame-sterilized forceps to a fresh BG11 agar plate containing appropriate antibiotics (section 8) to select for the cargo vector. Seal the plate with paraffin film.
Incubate the BG11 agar plate under appropriate growth conditions, as described above for Synechocystis PCC 6803 or S. elongatus UTEX 2973, until colonies appear. NOTE: Colonies typically appear after 7−14 days for Synechocystis PCC 6803 and 3−7 days for S. elongatus UTEX 2973.

5. Selection of conjugants

NOTE: Only cyanobacteria colonies carrying the cargo vector will be able to grow on the membrane.

Using a heat sterile loop, select at least two individual colonies from the membrane and streak onto a new BG11 agar plate containing appropriate antibiotics (Figure 5C). NOTE: Freshly streaked colonies may still be contaminated with E. coli carried over from conjugation (i.e., if small white colonies are evident on the plate), so two or three additional rounds of re-streaking onto fresh BG11 agar plates typically are needed to obtain an axenic cyanobacterial culture.
Confirm absence of E. coli contamination by inoculating a streak of cyanobacterial culture into a 15 mL centrifuge tube containing 5 mL of LB medium and incubating at 37°C overnight at 225 rpm in a shaking incubator. Following a sufficient growth period (~7 days), pick individual axenic colonies to set up liquid cultures for long-term cryostorage or subsequent experimentation.

Cyanobacteria colony PCR

Method 1

Materials

Cyanobacteria cells
DMSO
Thermocycler

Protocol

Resuspend a single colony in 10 μL DMSO.
Incubate 5 min at 95°C.
Use 1-1.5 μL of this mix as a template for PCR

Method 2

Materials

Cyanobacteria cells
Sterile water
-80°C freezer
60°C water bath
Thermoccycler

Protocol

Resuspend one colony in 10 μL sterile water.
Do 3 cycles of freeze (-80°C) and thaw (60°C) (5 min each).
Use 1 μL of this suspension as PCR template [For control reactions with plasmid templates, use 0.5 μL]

Monitoring growth culture

Materials

38°C incubator with shaking
LED panel for lighting
Flask 100 mL
BG11
Spectrophotometer

Protocol

All cultures were separated in 2 flasks: one in which all the samples were taken, and the other one only used to inoculate the following cultures. This allowed us to always have a non-contaminated culture because we did not work with it. OD monitoring was performed at 750 nm over several days, with regular sampling. The light intensity of the cultures was measured with a LI-COR lightmeter. The device was placed in the incubator to measure the illumination of the cultures.

Bioreactor

Protocol

Total volume of culture: 1.5 L

20 mL of culture flasks were inoculated with single colonies. These exponentially growing cultures were used to inoculate 200 mL of culture in 1 L baffled erlens. These erlen cultures were finally used to inoculate the reactors.
Samples were taken three times a day, the OD₇₅₀ was measured and HPLC vials were prepared (centrifugation 10 min at maximum speed and filtration of the supernatant with 0.45 micron filters). The HPLC analysis was Aminex HPX-87K Column. This potassium-form, 300 x 7.8 mm column and H₂O milliQ as mobile phase. to check the sucrose secretion.

For strain cscB:

BG11 media supplemented with 150 mM of NaCl
pH 7.5 (regulated with HCl and NaOH respectively)
Temperature 41°C
5 % CO₂
Light: Warm white light (LED) between 1500 μE (front of the reactor) and 200 μE (back of the reactor)
IPTG 1 mM (added in the reactors)
Kanamycin (10 μg/μL)

For strain cscB sps spp:

BG11 media
pH 7.5 (regulated with HCl and NaOH respectively)
Temperature 41°C
5 % CO₂
Light: Warm white light (LED) between 1500 μE (front of the reactor) and 200 μE (back of the reactor)

Sampling

The samples were collected with a syringe.
Sampling tube connects the bioreactor to the sampling chamber. Output tube connect the sampling chamber to the outlet, where the sample can be collected in a container. Aspiration tube connects the sampling chamber to the syringe.

To take a sample:

Unclamp the sampling and clamp the outlet tube.
Aspirate air with the syringe, this will fill the sampling chamber.
Clamp the sample tube and unclamp the output tube.
Evacuate the air from the syringe, causing the sample to flow out of the chamber through the output tube. The sample can be recovered at the outlet of the tube.

To collect samples:

Pinch the output tube and suck in air with the syringe.
Pinch the sampling tube, place a recipient tuve under the output tube to collect the sample and empty the air from the syringe.

Detection

HPLC

Sucrose

To analyze sucrose secretion, 1 mL of culture was collected and centrifuged at maximum speed for 5 minutes. The supernatant was collected and then filtered through a 0.2 μm filter. The sucrose secretion was quantified by HPLC with an Aminex HPX-87K Column This potassium-form, 300 x 7.8 mm column and mQ water as mobile phase. Column was kept at 70°C, flow rate 0.6 mL/min and analysis time 20 minutes.

Carotene

To quantify carotènes in our culture, 1 mL of culture was centrifuged and the supernatant eliminated. The pellet was resuspended in 1 mL acetone and the cells were broken with 500 μL of glass beads in a FastPrep-24™ Classic. The mechanical lysis conditions were: one cycle of 40 s of shaking at 6m/s. After centrifugation, the supernatants were analyzed on a C18 RP HPLC column,Thermo Scientifc™ Hypersil C18 GOLD™, 3 μm, and 2.1×100 mm column, with a mixture of acetonitrile:methanol:isopropyl (85:10:5 v/v) as eluant. Column was kept at 20°C, flow rate 0.5 mL/min and analysis time 15 minutes.

GC-MS

The analyses were performed on a GC-MS Trace 1310-ISQ (Thermofisher) equipped with a single quadrupole mass spectrometer with Helium as carrier gas. The column used was a ZB-1701 (Phenomenex - 7HG-G006-05-C) with the following dimensions: Length: 30 m, Internal diameter: 0.25 mm, Film thickness: 0.15 mm).

The analysis conditions are listed in the followed table:

Parameters	Unity	Values
Injector temperature	[°C]	250
Mobile phase flow	Mobile phase flow [mL.min^-1]	0.9
Oven temperature gradient	[°C.min^-1]	7, 4, 25
Transfer tube temperature	[°C]	250
Source temperature	[°C]	300
m/z range followed	[/]	35 to 700

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