Feb 10, 2025

Public workspaceAutomated combinatorial media preparation V.2

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.81wgbx7eylpk/v2
  • 1Lawrence Berkeley National Laboratory;
  • 2Joint BioEnergy Institute;
  • 3Agile BioFoundry;
  • 4Basque Center for Applied Mathematics
  • JBEI
Matthew Incha
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DOI: dx.doi.org/10.17504/protocols.io.81wgbx7eylpk/v2
Protocol Citation: Tijana Radivojevic, Matthew Incha, Vincent Blay, Apostolos Zournas, Stephen Tan, Hector Garcia Martin 2025. Automated combinatorial media preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx7eylpk/v2Version created by Matthew Incha
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 17, 2023
Last Modified: February 10, 2025
Protocol Integer ID: 119925
Keywords: automated media prep, media optimization, combinatorial media
Funders Acknowledgements:
Hector Garcia Martin
Grant ID: Department of Energy DE-AC02-05CH11231
Hector Garcia Martin
Grant ID: Basque Center for Applied Mathematics CEX2021-001142-S
Abstract
Although synthetic biology can produce valuable chemicals in a renewable manner, its progress is still hindered by a lack of predictive capabilities. Media optimization is a critical, and often overlooked, process which is essential to obtain the titers, rates and yields needed for commercial viability. Here, we present a molecule- and host-agnostic active learning process for media optimization that is enabled by a fast and highly repeatable semi-automated pipeline. Its application yielded 60% and 70% increases in titer, and 350% increase in process yield in three different campaigns for flaviolin production in Pseudomonas putida KT2440. Explainable Artificial Intelligence techniques pinpointed that, surprisingly, common salt (NaCl) is the most important component influencing production. The optimal salt concentration is very high, comparable to seawater and close to the limits that P. putida can tolerate. The availability of fast Design-Build-Test-Learn (DBTL) cycles allowed us to show that performance improvements for active learning are rarely monotonous. This work illustrates how machine learning and automation can change the paradigm of current synthetic biology research to make it more effective and informative, and suggests a cost-effective and underexploited strategy to facilitate the high titers, rates and yields essential for commercial viability.

Here we provide the protocol for automated media optimization on a flaviolin producingPseudomonas putida KT2440 strain.
Protocol materials
ReagentFilter-sterilized HPLC-grade water
Step 4
ReagentIron (II) sulfateFisher ScientificCatalog #7782-63-0
Step 4
Safety warnings
Wear PPE.
Before start
Make sure you booked the equipment in the calendar:
  • 978-PRO-BIOLECTOR (4148) EQ and
  • 978-4-BIOMEK-NX-S8_ SPECTRAMAX (4148) EQ (1) or
  • 978-4-BIOMEK-NXP (4148) EQ (1)

Make sure you received proper training before operating on the Biomeks and Biolector.

Have an overnight of your culture ready.

Required equipment/labware
Required equipment/labware
Destination plate:

m2p flower plate
(for running in the Biolector)
m2p black bottom flower plate
(for running off the Biolector)

Water plate:
deep reservoir (available at Robotics lab)

Source plate (with stock components):
24-deep well x3
Liquid handler:
Equipment
new equipment
NAME
Beckman Coulter
BRAND
Biomek NXp
SKU

Pipette tips needed (available at Robotics lab):
  • tips f200 (light green box)
  • tips f20 (light blue box)
Fermentation platforms available:
  • [Biolector]
  • Biolector Pro or
  • Multitron
Additional components:
  • ReagentFilter-sterilized HPLC-grade water
  • Kanamycin

To be prepared fresh for every cycle:
  • ReagentIron (II) sulfateFisher ScientificCatalog #7782-63-0
  • Overnight culture of P. putida PP_5404::attB::pIS100. (The base strain for this assay was simply KT2440 modified for PhiC31 integrations.)


Additional labware:
  • 5mL, 10mL pipettes
  • 3x 50mL tubes for FeSO4
  • filter, syringe for FeSO4 sterilization
  • 2x 2mL tubes for culture

Additional equipment:
  • vortex
  • centrifuge
  • flame
Calculate stock concentrations
Calculate stock concentrations
Input:
  • total volume of the culture (we use Amount1500 µL as total volume)
  • standard media recipe (e.g. Download standard_recipe_concentrations.csvstandard_recipe_concentrations.csv )

Run the notebook A_Find_Stock_Concentrations.ipynb.
Prepare stock solutions
Prepare stock solutions
Use the list of components concentrations created in step 5 and prepare stock solutions. Ideally, you should prepare enough volume for all DBTL cycles.
Note
Steps 5 and 6 should be performed only once during a study and not for every DBTL cycle.

Create stock plates definitions
Create stock plates definitions
Run the notebook B_Create_Stock_Plates.ipynb. Prepare the stock plates following the instructions resulting from the notebook by aliquoting the components into the appropriate wells of the source plate. A stock plate with culture and FeSO4 should be prepared fresh for every cycle. The other two, with high and low level concentrations, can be used and refilled if needed for consecutive cycles.

Example output files:
  • source plate instructions Download 24-well_stock_plate_high.csv24-well_stock_plate_high.csv Download 24-well_stock_plate_low.csv24-well_stock_plate_low.csv Download 24-well_stock_plate_fresh.csv24-well_stock_plate_fresh.csv

An example of how to prepare a stock of Kanamycin:
  • Take 1000x concentration from the freezer
  • If we need e.g. Amount1 mL of the 300x concentration in the stock plate, we need a 3.33 dilution, so Amount300 µL of Kan and 1000-300=Amount700 µL of H2O
Choose target media composition
Choose target media composition
E.g. by using ART. For the initial cycle you may run the notebook C_Initial_Media_Designs.ipynb. Once there is a training data set you may run the notebook C_ART_Media_Designs.ipybn.

Example output file: Download target_concentrations.csvtarget_concentrations.csv
Create files for biomek transfers and source plate instructions
Create files for biomek transfers and source plate instructions
Run the notebook D_Create_Transfers.ipynb. It will generate a file with volumes for all components for all wells, files for biomek that are used to define transfers of those volumes, and stock plate definitions with additional columns with required volumes for each stock component, for this particular run.

Example output files:
  • destination volumes Download dest_volumes.csvdest_volumes.csv
  • source plate instructions Download 24-well_stock_plate_high.csv24-well_stock_plate_high.csv
  • biomek files Download P20_components.csvP20_components.csv Download P20_culture.csvP20_culture.csv Download P20_kan.csvP20_kan.csv Download P20_water.csvP20_water.csv Download P200_components.csvP200_components.csv Download P200_water.csvP200_water.csv

This notebook will also provide a number of tip boxes needed to perform the transfers. Most likely, the number of boxes will be larger that the number of slots on the Biomek deck, so the method will need to pause, which gives you time to refill the deck with tips.
Prepare the source plates
Prepare the source plates
Make sure the stock plates with low and high concentration levels, prepared at step 7, have at least volume levels as defined in step 9.
Safety information
Perform this step in a sterile environment (use flame).
Prepare the plate with fresh stocks:

FeSO4:
  • To prepare Amount20 mL of Concentration60 millimolar (mM) stock, find molar mass of the iron-sulfate available and use a calculator to find mass in mg
  • Typical stock is FeSO4*7H2O aka the heptahydrated salt. 333.612mg heptahydrate / 20mL sterile H2O to make 60mM stock
  • Measure and add FeSO4*XH2O into a 50 mL falcon tube, add Amount20 mL of H2O, vortex
  • The solution may be cloudy before the filtration. This is because of some oxidized FeSO4 (new insoluble Fe(III) species) in the solution. Do not be alarmed. This is a small proportion of the stock iron sulfate that was measured.
  • Use a sterile 0.2uM luer-lock syringe filter (or SteriFlip if available) to filter sterilize the 60mM solution into a new 50mL conical tube
  • Find volume of this solution needed to create 10 mL of solution with target concentration of Concentration0.3 millimolar (mM) using the formula:
i.e. 10mL * 0.3mM / 60mM = 0.05 mL of FeSO4
  • Add Amount9.95 mL of H2O to another (falcon) tube and Amount0.05 mL of FeSO4, vortex, filter sterilize


Culture:
  • Take 2mL of overnight culture and place into the 2mL tube (2x)
  • Centrifuge the 2 2mL tubes of culture at Centrifigation10000 rcf, 00:02:00
  • Decant the supernatant
  • Re-suspend the overnight culture with an equal volume MOPS minimal medium without carbon
  • Place the resuspended culture in the appropriate well of the source plate
2m
Biomek setup
Biomek setup

Note
If you wish to set up a method in advance of a run, you may access the Biomek remotely.

  • Follow the instruction provided in this file.
  • Request a password for a Windows Active Directory (AD) account from Arthur Panganiban (ahpanganiban@lbl.gov)
  • Find your Biomek, use password Robotp@ss978

Open the Biomek Software application on the Biomek. At the top toolbar, select Project > Open Project to open the "Automation Dev" project.

At the top toolbar of the Biomek Software, select File > Open to open the method "comb_media_48BIOLECT_V5"

Set up the deck as specified by the Instrument Setup pop-up. Check if the number of tip boxes corresponds to what was calculated in step 9, notebook D_Create_Transfers.ipynb.

Check that the deck layout after the pause (second "Instrument Setup" step) corresponds to the number of tip boxes calculated in Step 9, notebook D_Create_Transfers.ipynb.


For each of the "Transfer From File" steps, upload the corresponding Biomek files created in Step 9.
For example:

Biomek simulation
Biomek simulation
To run the Biomek in the simulation mode choose the following:




Biomek run
Biomek run

Note
Do not start a Biomek run remotely if it is not in the Simulation mode.

Hit the play button (green arrow) to run the method.

Note
Visually inspect if aspirations are happening and there is nothing suspicious. At the end inspect if all the wells have the same total volume.

During the run you may touch the light curtain to pause the Biomek. The following window will pop-up.


Biolector/Multitron run
Biolector/Multitron run
2d
2d

Note
Before operating the BioLector get appropriate training.

On the Biolector, open the protocol labelled "matthew_flower".

Adjust the calibration "lot number" to that which corresponds to the sticker on the M2P biolector flower plate.

If the lot number is unavailable, import it and load it into the matthew_flower method.
(you should have been trained on this. if you can't remember how, email the current biolector superuser)

Place the plate inside the machine and start the method.

Temperature Temperature30 °C
Humidity 80%
Length of the run Duration48:00:00
Shake Speed (800 rpm)
Filters:
  • ID401 (620nm for biomass measurement)
  • ID402 (pH measurement)
  • ID403 (DO measurement)
2d
On Multitron:
Temperature Temperature30 °C
Shake speed 700RPM

Place plate in Infors Multitron


Absorbance assay
Absorbance assay
Follow a protocol for absorbance assay from an m2p plate.
Upload data into EDD
Upload data into EDD
Run the notebook E_Create_EDD_Study_Files.ipynb.

Once you create files for EDD import, follow a protocol for EDD study creation and a protocol for importing data into EDD.