Feb 10, 2025

Public workspaceAbsorbance assay from an M2P flower plate V.2

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.x54v9pr51g3e/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.x54v9pr51g3e/v2
Protocol Citation: Tijana Radivojevic, Matthew Incha, Apostolos Zournas, Vincent Blay, Stephen Tan, Hector Garcia Martin 2025. Absorbance assay from an M2P flower plate. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pr51g3e/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: September 18, 2023
Last Modified: February 10, 2025
Protocol Integer ID: 119927
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.

This protocol explains the steps for preparing samples and taking OD600 and OD340 measurements from a BioLector plate.


Safety warnings
Wear proper PPE.
Before start
Make sure you booked the equipment in the calendar:
  • 978-4-BIOMEK-NX-S8_ SPECTRAMAX (4148) EQ (1) (or any other "plate-reader" capable of measuring OD340 and OD600)
  • 978-4-BIOMEK-NXP (4148) EQ (1)

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


Required equipment/labware
Required equipment/labware
Destination plate:

Flat-black clear-bottom Tecan plate x2

Water plate:
deep reservoir (available at Robotics lab)
Supernatant plate:
96-deep-well plate x2

Liquid handler:
Equipment
new equipment
NAME
Beckman Coulter
BRAND
Biomek NXp
SKU

Pipette tips needed (available at Robotics lab):
  • tips s200 (green box)
  • tips p1000 (yellow box)

Spectrophotometer/plate-reader:
  • Molecular Devices SpectraMax M2
  • or any other spectrophotometer capable of measuring OD340 and OD600

Additional components:
  • H2O
  • m2p plate with cultures

Centrifuge options:
  • Avanti-15R-centrifuge
  • Allegra-25R
  • Eppendorf 5810R
  • (any swinging-bucket centrifuge capable of spinning a 96-deepwell plate)
  • NOT the Eppendorf 5430


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

We will use two Biomek methods for this assay.

The first method will prepare:
  • a Tecan plate for OD600 measurements, with 10x dilution of the samples
  • two 96-deepwell plates as an intermediate step for preparing a Tecan plate for OD340 measurements - one with 1 mL of samples (supernatant plate) and the other with 1 mL of water for balance in the centrifuge

The second method will prepare:
  • a Tecan plate for OD340 measurements (aliquoting cell supernatants from the 96-deepwell plate)
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 first method.

To open the second choose:


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.


  • The first method takes around Duration00:05:00 before the pause and Duration00:05:00 after the pause
  • When the method pauses, remove the two 96-deepwell plates and continue the Biomek run
10m
Centrifuge run:

  • Place the two 96-deepwell plates sealed in the centrifuge and spin at max speed for Duration00:05:00
5m
  • Carefully remove the supernatant plate from the centrifuge, remove the seal and place it back in the Biomek
  • Start the second Biomek method to prepare a Tecan plate for OD340 measurements (takes around Duration00:05:00 )

5m
SpectraMax run
SpectraMax run
  • Start the Spectramax software "SoftMax"
  • Click "Open a data file" in the top left of the SoftMax window
  • Open the method for measuring OD600 named OD600.sda in the "My_Documents/Keasling_Lab/inch" subfolder on the Spectramax (NX-S8) computer


  • Open tray and place plate
  • Measure OD600 from the first prepared 96-well Tecan plate
  • Save the data (see step 7)

  • Click "Open a data file"
  • Open a method for measuring OD340 named OD340.sda in "My_Documents/Keasling_Lab/inch" subfolder on the Spectramax (NX-S8) computer
  • Measure OD340 from the second prepared 96-well Tecan plate
  • Save the data (see step 7)


Saving data
Saving data
  • Copy the values for OD600 from the SoftMax window into an empty Excel file and rename the spreadsheet to "600"
  • Copy the values for OD340 to another spreadsheet of the same file and rename to "340"
  • Save the Excel file as "OD.xlsx"