Apr 15, 2025

Public workspaceA Modular High-Throughput Pipeline for Automated Microbial Strain Engineering: From Colony Picking to Phenotypic and Genotypic Analysis V.1

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DOI
dx.doi.org/10.17504/protocols.io.261gerqkdl47/v1
  • 1Lawrence Berkeley National Laboratory;
  • 2Joint BioEnergy Institute;
  • 3Agile BioFoundery
  • Agile BioFoundry
  • LBNL
Rita Kuo
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DOI: dx.doi.org/10.17504/protocols.io.261gerqkdl47/v1
Protocol Citation: Rita Kuo, Randy Louie, Jennifer Gin, Sam Yoder, Stephen Tan, Nathan J Hillson, Christopher J Petzold 2025. A Modular High-Throughput Pipeline for Automated Microbial Strain Engineering: From Colony Picking to Phenotypic and Genotypic Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerqkdl47/v1
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: March 11, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 124230
Keywords: High-throughput, automation, liquid handler, Hamilton, metabolic engineering, strain engineering, robotics, Large-scale microbial sample preparation
Funders Acknowledgements:
Department of Energy (DOE) Office of Science
Grant ID: DE-AC02-05CH11231
Department of Energy (DOE) EERE-BETO Agile BioFoundry (ABF)
Grant ID: DE-AC02-05CH11231
Abstract
Microbial strain engineering has evolved to incorporate guide RNA, oligo pools, and random insertion libraries with millions of combinations to decipher mechanisms of bioproduct production in host organisms. However, effective high-throughput screening of individual colonies for both genotypic and phenotypic traits remains challenging.

We present an integrated high-throughput automation pipeline capable of handling over 5,000 samples daily for comprehensive strain engineering tasks. This system bridges the gap from initial colony selection through genotyping and phenotypic analysis using multiple analytical platforms.

Our modular workflow consists of several integrated processes:
  1. High-throughput colony picking using automated colony picking systems
  2. Media preparation and distribution with customizable volumes and plate formats
  3. Glycerol stock and production plate preparation with flexible options for culture transfer, replication, and preservation
  4. Culture transfer for scaled production allowing for additional stamping operations with customizable media compositions
  5. Sample preparation for genotyping and phenotyping for specialized assays (proteomics, RapidFire) and NGS phenotyping with configurable transfer parameters

This pipeline offers significant flexibility, allowing researchers to employ individual modules independently or reconfigure workflows according to specific experimental requirements. The system's standardization enhances reproducibility while accelerating strain engineering cycles for synthetic biology and bioproduction applications.
Image Attribution
Created in Biorender Petzold, C. J. 2025.




Guidelines
This protocol is designed for processing high-throughput samples (n > 1,000) for applications such as glycerol stocks, phenotyping (e.g., Plate reader, RapidFire assays, and/or proteomics), and genotyping (e.g., Colony PCR, barcode sequencing). Potential applications include RB-Tn libraries, oligo pool libraries, Cas9 editing libraries, and more.
Materials
Instruments
Hamilton Vantage with Plate Sealer, Plate Pealer and Plate Reader (Hamilton Company, Reno, NV, United States)
Qpix 460 Colony Picker (Molecular Devices, CA, United States)
Eppendorf Centrifuge 5810R and (Eppendorf, CT, United States)
Agilent PlateLoc (Agilent, CA, United States)
Vortex-Genie 2 (Scientific Industries, Inc. NY, United States)


Reagents (dependent on experimental conditions)
Growth media (e.g., LB, M9)
Inducers (e.g., IPTG)
Selection Markers (e.g., Kanamycin)

Consumables
Tips (Hamilton Company, Reno, NV, United States)
96 deep-well Plates (Beckman Coulter, Brea, California, United States)
96-well round-bottom Nunc Plates (Thermo Fisher Scientific, Waltham, Massachusetts, United States)
Shallow or Deep Reservoirs (Thermo Fisher Scientific, Waltham, Massachusetts, United States)
Corning Coast Assay Plates, Black with Flat Bottom ( Corning Inc. Maine, United States)

Safety warnings
- Wear proper PPE (gloves, safety goggles, and lab coat), and prepare solvents in a chemical fume hood.
- Store organic solvents in a flammable storage cabinet when not in use.
- Discard used materials, solvents, and buffers in appropriate waste containers according to your institution's procedures.
Before start
Sample tracking: It is important to establish proper sample tracking before initiating any set of high-throughput experiments. We recommend barcoding according to standard LIMS guidelines to minimize sample tracking issues.

Plate sealer: If you want to seal your plates, configure and test the sealer parameters for optimal sealing. The ideal temperature and time settings vary depending on the specific plate material and seal type. It is recommended to consult the manufacturer's guidelines and conduct preliminary tests to determine the optimal settings. The goal is to achieve a seal that is easy to peel while maintaining a firm seal for each well. Monitor the sealing process to ensure the desired balance between peelability and seal integrity.
Grow Colonies on Agar plates
Grow Colonies on Agar plates
1d
1d
Scale: 5-10 agar plates

Before you start:
Sterilize your bench and equipment before handling the cultures.





Input materials: Glycerol stocks that contain mutant libraries and agar plates
Output: Agar plates with well-separated colonies


Steps:
1. Thaw your cultures on ice and perform serial dilution (1X, w102X, 103X, 104X, 105X) in 1.5 mL tubes.
2. Transfer 5 µL of the culture dilutions to the Petri dish and spread the culture gently using glass beads. For Q-trays, use 20 µL of the culture dilutions and spread the culture gently using glass beads.
3. Place the plates in a sterilized environment to let it dry if necessary.
4. Once dry, place the plates upside down at the optimal growth temperature to let the culture grow until the colonies reach a diameter of at least 1 mm.

Note
1. Do not over dry the plate. This requires careful monitoring of the process multiple times until the drying rate is well established. Utilization of a controlled drying environment is highly recommended.
2. Use the appropriate growth media for your strain when preparing culture dilutions.
3. Colony outgrowth time may vary from 12 hours to several days, depending on the strain.

30m
Pick Colonies and Grow Cultures in Nunc Plates
Pick Colonies and Grow Cultures in Nunc Plates
3h
3h
Pre-fill Nunc plates with media

Scale: 16 plates



Before you start
Perform a pilot test to determine culture outgrowth conditions (e.g., temperature, shaking speed, incubation time) in round-bottom Nunc plates.

Prepare Nunc plates on Hamilton Vantage
Source plates: Deep-well reservoir
Destination plates: 96-well Nunc Plates (round bottom)
Tip type: 1000 µL Filtered Tips
Input materials: Nunc Plates and growth media
Output: Barcoded Nunc Plates with 150 µL of growth media per well

Steps:
  1. Place barcodes or labels on the Nunc Plates
  2. Transfer 150 µL of growth media per well from the reservoir to the 96-well Nunc plates using the same set of 300 µL tips for all destination plates.
  3. Examine the plates and briefly centrifuge those plates that contain bubbles.
  4. Proceed with colony picking immediately to minimize evaporation and chances of contamination.

Note
1. If antibiotics or other methods of selection are needed, be sure to include the necessary components in the media.
2. If you are starting from a diverse genomic library of strains it is good practice to validate your library at this step to identify any potential bias in library composition.





30m
Prepare positive controls
Scale: 16 X 96-well Nunc plates

Before you start:
  1. Grow 5 mL of positive control strains (or your base strains) until the culture reaches an OD₆₀₀ of approximately 0.6.
  2. Mix well, then aliquot 150 uL of the culture into 96-well Nunc plates, ensuring at least three wells are used for each strain.

Prepare positive controls for each Nunc plate using the Hamilton Vantage
Source plates: A 96-well Nunc plate containing positive control strains in growth media, with at least 3 different replicates for each strain
Destination plates: 96-well Nunc Plates containing growth media
Tip type: 50 µL Filtered tips
Input materials: A 96-well Nunc plate containing positive control strains in growth media and 96-well Nunc plates containing growth media
Output: 96-well Nunc plates containing transferred positive control strains

Steps:
1. Mix the plate containing positive controls by pipetting 50  µL of the culture mixture up and down at least 10 times using 50  µL tips.
2. Lift the tip and dip it into the corresponding well of a Nunc plate containing growth media.
3. Mix the growth media by pipetting 50  µL up and down 5 times.
4. Repeat steps 1–3 for each set of positive control and growth media plates until all Nunc plates are processed.

30m
Pick colonies with Qpix automated colony picker

Scale: 16 X 96-well Nunc plates containing up to 1,536 microbial strains




Before you start
Design plate maps on Qpix to reserve wells for positive controls (e.g., baseline strains) and negative controls (e.g., wells containing only growth media).

Pick colonies using Qpix
Source plates: Q-trays or Petri dishes
Destination plates: 96-well Nunc Plates
Input materials: Agar Petri dishes or Q-trays containing colonies; barcoded Nunc plates with 150 uL of growth media per well
Output: Nunc plates with a single colony in each well

Steps:
Set up Qpix
  1. Fill each corresponding wash tray with 0.1% bleach, nuclease free water and 80% Ethanol.
  2. Set the UV light on the QPix to run for 10 minutes using the QPix Colony Picking Software.
  3. Check if the 96-picking pin head is installed. If not, proceed with the following step.
  4. Open the Qpix control program. Go to "Instrument Utilities", click "Change head" to install picking head. Screw the knob tightly to hold the head in place.
  5. Perform "Camera Alignment Process" to calibrate the imaging camera with the newly mounted picking head.

Pick colonies
1. Use the QPix Colony Picking Software. In the navigation window, double-click the “Picking” icon to begin.
2. Follow the instructions to set up your picking routine or select a saved file.
3. Select destination stackers by clicking th yellow rectangle on the screen and click Next.



4. Set up the head and sanitization conditions in the Head/Sanitise window. It is recommended to use at least 5 wash cycles, with 5 seconds of dry time and wait time each.


5. Choose your picking source in the "Source" window and set the picking depth into the agar. (The number of plates may vary depending on the number of pickable colonies and the total number of colonies you wish to pick.)


6. In the Destination Options, set the number of dips as needed. Then, under the Destination Microplate Template, click “Edit” to deselect the wells assigned to your negative controls and baseline strains. QPix will skip the wells that have been opted out during colony picking.



7. Review the setting summary after the Settings Summary window appears.
8. Load the Nunc culture plates with 150 µL of LB containing suitable antibiotics per well into the stacker holder.
NOTE: Destination plates must be oriented with A1 in the front right corner; otherwise, the picking logs will misrecord the actual well mappings.
9. Place the stacker holder into position and turn knob to lock.
10. Place Q-trays or Petri dishes on the holder.
11. Run the program.
13. After the picking process is done, remove Nunc plates from the holder.
14. Seal the Nunc plates with air permeable seals and incubate the plates to let the culture grow to at least OD₆₀₀ ~ 0.6.

Clean up Qpix
  1. Take the wash trays 1-3 out of the picker and empty wash tray one and two into the sink, wash tray with 80 % ethanol must be poured into the SAA for flammable solvents.
  2. Rinse all three wash trays with water and place back into the picker.
  3. Set the UV decontamination to run for 10 minutes using the QPix Colony Picking Software.


Note
Key parameters to adjust on Qpix:
A. Number of dips
B. Inoculation height
C. Picking depth into agar
D. Number of source plates (This depends on the number of pickable colonies per plate and the target number of colonies for your experiment)
E. Plate source (Petri dish or Q-tray)
Kay parameters to adjust on culture outgrowth
A. Incubation time
B. Incubation temperature
C. Shaker speed

It is recommended to grow cultures in an incubator with humidity control to minimize evaporation.

2h
Prepare 96 Deep-Well Plates for Production Outgrowth
Prepare 96 Deep-Well Plates for Production Outgrowth
40m
40m
Pre-fill deep-well plates with media
Scale: 64 X 96-deep well plates



Before you start
If you want to seal the induction plate for short-term storage, configure the sealer parameters for optimal sealing.

Fill the 96 deep-well plates with production media on the Hamilton Vantage
Source plates: Reservoir
Destination plates: 96 deep-well plates (2 mL)
Tip types: 1000 µL Filtered tips
Input materials: induction media and 96 deep-well plates
Output: Induction plates containing growth media only

Steps:
1. Place barcodes or labels on the south side of the plate.
2. Transfer 800 µL of induction media per well from reservoir to deep-well plates with the same set of tips.
3. Optional: seal the plate for short-term storage


Note
1. Include antibiotics and other required inducers as needed for your experiment. 2. It is recommended to use 800–1000 uL of induction media in 96 deep-well plates. This volume helps minimize evaporation while avoiding splashing and cross-contamination during incubation.
3. Prepare extra plates and media to ensure flexibility during setup and to accommodate any last-minute changes or sample loss during the process.



40m
Prepare Glycerol Stocks
Prepare Glycerol Stocks
1h 30m
1h 30m
Scale: 16X 96-well Nunc plates
Before you start
1. Examine the Nunc plates to ensure culture outgrowth; an OD600 ≥ 0.6 is recommended.
2. Configure the sealer parameters for optimal sealing.




Prepare glycerol stock on the Hamilton Vantage
Source plates: A deep-well reservoir that contains 240 mL of 60 % glycerol
Destination plates: Nunc Plates with a single colony outgrowth in each well
Tip types: 300 µL tips
Input materials: 60 % glycerol; Nunc Plates with culture outgrowth, foil seals for -80 °C storage
Output: Sealed Nunc Plates with glycerol stocks

Steps:
  1. Perform a quick spin (<10 seconds) to remove any liquid from the seal, then carefully peel the Nunc plates to avoid cross-contamination (this step is optional for the 96-deep-well plates described in the previous step).
  2. Using 300 uL tips, transfer 50 uL of 60% glycerol to each well of the Nunc plate containing 150 uL of culture.
  3. Using the same tips, mix 100 uL of the glycerol-culture mixture by pipetting up and down 10 times.
  4. Discard used tips and use a fresh set of tips for each new source plate.
  5. Seal the plates with a foil seal—unless you are proceeding directly to induction plate preparation.
  6. Store the plates at -80 °C or continue with induction plate preparation.

Note
Key parameters to adjust on the sealer:
A. Seal temperature
B. Seal time


1h 30m
Transfer Cultures for Production Cultures
Transfer Cultures for Production Cultures
2h 30m
2h 30m
Scale: 64 X 96-deep well plates containing 4 replicates for each strain (6,144 samples)

Inoculate culture from Nunc plate to 96-deep well plates with replicates




Before you start
Configure the sealer parameters for optimal sealing.

Note
Key parameters to adjust on the sealer:
A. Seal temperature
B. Seal time



Prepare induction plates on the Hamilton Vantage
Source plates: 96-well Nunc Plates
Destination plates: 96 deep-well plates
Tip type: 50  µL Filtered tips
Input materials: Glycerol stock plates (96-well Nunc Plates), induction plates containing growth media only
Output: Induction plates with single strain in each well

Steps:
1. Label the 96-well induction plates with barcodes or plate IDs.
2. Transfer 5 uL of stock culture per well into the induction plates.
3. Repeat step 2 until all replicate plates are prepared.
4. Discard used tips and use a fresh set of tips for each new source plate.
5. Seal the induction plates with permeable seals.
6. Incubate the induction plates under appropriate conditions.
7. Store the stock culture plates at -80 °C for future use.

Note
1. It is recommended to keep the transfer volume between 5–10 uL to reduce the risk of contamination and to maintain better control over the growth phase for downstream experiments. Transferring a large amount of bacteria may cause some strains to reach stationary phase within a few hours.
2. For high-throughput work, at least three replicates are important to produce reliable phenotyping data.

Key parameters to adjust on induction: It is good practice to perform a pilot test on a single plate to validate the following conditions:
A. OD₆₀₀ of your stock culture B. Transfer volume on the robot C. Culture outgrowth time (some experiments collect samples during the log phase, while others require stationary phase cultures)




2h 30m
OD₆₀₀ Measurement or Fluorescent Detection
OD₆₀₀ Measurement or Fluorescent Detection
3h 30m
3h 30m
Scale: 64 X 96-well plates (6,144 samples)

Before you start
Set up parameters on Soft Max Pro for your project.

Note
1. It's important to determine the linear range of the measurement. Performing a pilot test before setting up high-throughput work is recommended.
2. Consider to include standards for a standard curve for better quantification in your run as needed.

Key parameters to consider for culture outgrowth:
  1. Data collection timing
  2. Optimal cell density or fluorescence intensity

Key parameters to adjust on the plate reader via SoftMax Pro:
1. For OD₆₀₀ measurements :
A. Read mode: ABS (absorbance)
B. Read type: Endpoint
C. Wave length: 600 nm
D. Shaking before first read: enable and set for at least 10 seconds
2. For fluorescence detection: A. Read mode: FL (fluorescence)
B. Read type: Endpoint
C. Excitation and emission wavelengths: users' choice
D. Shaking before first read: enable and set for at least 10 seconds






Perform automatic plate reading on the Hamilton Vantage

Source plates: 96-deep-well induction plates containing single-strain culture outgrowths
Destination plates: Clear-bottom plates compatible with plate readers
Tip types: 50  µL and 300  µL Filtered tips
Input materials: 96-deep-well induction plates containing single-strain culture outgrowths and clear-bottom plates compatible with plate readers
Output: OD₆₀₀ or fluorescent data

Option 1: Without Dilution
(Only 300 µL tips are required)
  1. Label the 96-well reading plates with barcodes or plate IDs.
  2. Mix the induction plates by pipetting 100 µL of the culture mixture up and down at least 10 times using 300 µL tips.
  3. Transfer 180 µL of the culture mixture per well to the reading plates using the same tips.
  4. Discard used tips and use a fresh set of tips for each new source plate.
  5. Place the reading plate into the plate reader.
  6. Measure OD₆₀₀ or fluorescence on plate reader.

Option 2: With Dilution

(May require both 50 µL and 300 µL tips depending on your dilution factor)
  1. Label the 96-well reading plates with barcodes or plate IDs.
  2. Add diluent per well to the reading plates according to your dilution factor using the same set of tips for all the destination plates.
  3. Discard the used tips after diluents have been added to all plates.
  4. Mix the induction plates by pipetting 100 µL of the culture mixture up and down at least 10 times using 300 µL tips.
  5. Transfer the appropriate volume of culture mixture per well to the reading plates using the same set of tips.
  6. Discard used tips and use a fresh set of tips for each new source culture plate.
  7. Place the reading plate into the plate reader.
  8. Measure OD₆₀₀ or fluorescence on plate reader.

Note
1. If you don't have a plate reader integrated into your automation system, consider preparing plates with your liquid handler and reading them on a standalone plate reader.
2. You may want to dilute your samples prior plate reading if the cell density or fluorescent is too high. In many cases, it's good to keep OD₆₀₀ between 0.1 -1.
3. It's more efficient if you can avoid diluting your samples. This can be achieved by performing a pilot test to identify the appropriate cell density or fluorescent intensity under certain growth conditions. However, in some cases—such as when collecting samples in stationary-phase cells is desired—the culture may exceed the optimal range for measurement and dilution will be necessary before plate reading.

Key parameters to adjust on your liquid handler:
Dilution factor for your samples


3h 30m
Process Plates for Genotyping and Phenotyping
Process Plates for Genotyping and Phenotyping
6h 30m
6h 30m
Scale: 16 X 96-well plates

Collect samples for genotyping

Source plates: 96 deep-well plates or 96-well Nunc plates
Destination plates: 384-well plates
Tip type: 50  µL Filtered tips
Input materials: 96 deep-well plates or 96-well Nunc plates containing single-strain culture mix and 384-well plates
Output: 384-well plates containing single-strain cell mixtures

Steps:
  1. Label the 384-well plates with barcodes or plate IDs.
  2. Transfer 20 µL of cell mixtures from each well of four 96-well plates and combine them into a single 384-well plate.
  3. Discard used tips and use a fresh set of tips for each new source culture plate.
  4. Repeat Step 2 and 3 until all plates are processed.
  5. Seal the 384-well plates and store them at –20 °C for preservation.

30m
Collect samples for RapidFire-MS assays
Scale: 64X 96-well plates



Source plates: 96 deep-well plates
Destination plates: 96-well-full-skirted plates
Tip type: 300  µL Filtered tips
Input materials: 96-deep-well induction plates containing single-strain culture outgrowths and 96-well-full-skirted plates
Output: 96-well-full-skirted plates with supernatant that contain bioproducts

Steps:
  1. Label the 96-well full-skirted plates with barcodes or plate IDs.
  2. Centrifuge the 96-deep-well induction plates at 3,000 × g for 5-30 minutes to pellet the cells (see Note below).
  3. Transfer 10-100 µL of the supernatant from the 96-deep-well plate into 96-well full-skirted plate (see Note below).
  4. Discard used tips and use a fresh set of tips for each new source plate.
  5. Seal the full-skirted plates with pierceable foil seals.
  6. Store the sealed plates at –20 °C for future use.


Follow RapidFire-MS sample preparation protocol for the target molecule of interest, for example:
High-throughput quantitative analysis of isoprenyl acetate (IPA) by using an Agilent RapidFire-QqQ system


Note
You may want to consider adding an additional filtration step before performing the RapidFire assays to ensure removal of particulates that could interfere with MS analysis.


Key parameters
A. Centrifugation time: This is dependent on the organism and the growth conditions of your cultures.
B. Transfer volume: This is dependent on the amount of the target molecule in your cultures and the sensitivity of your HTS assay. Additional dilution steps may be required for target molecules that are present at high concentration in the culture (e.g., >1.0 g/L).


3h 30m
Collect samples for proteomics on Vantage

Scale: 16X 96-well plates



Source plates: 96 deep-well plates
Destination plates: 96-well unskirted plates
Tip types: 300  µL and 1000  µL Filtered tips
Input materials: 96-deep-well induction plates containing single-strain culture outgrowths and 96-well unskirted plates
Output: 96-well non-skirted plates with cell pellets

Steps:
  1. Label the 96-well non-skirted plates with barcodes or plate IDs.
  2. Centrifuge the 96-deep-well induction plates at 3,000 g for 5 minutes.
  3. Use 1000  µL tips to remove 600  µL of the supernatant per well.
  4. Mix the culture at least 10 times with the following parameters set up on the robot:
  • Mix volume: 200  µL
  • Aspirate height: 0.2 mm
  • Dispense volume: 150  µL
  • Dispense height: 1 mm
5. Discard used tips and use a fresh set of tips for each new source culture plate.
6. Centrifuge the non-skirted plates at 4000 rpm for 3 minutes.
7. Remove the supernatant from the non-skirted 96-well plate by inverting or aspirating.
8. Seal the 96-well non-skirted plates with foil seals.
9. Store the stock culture plates at -80 °C for future use.


Note
It is recommended the culture reach OD₆₀₀ at least 0.8. You may want to test a few samples to verify minimum OD₆₀₀ and the volume of cell culture for proteomics. You will need enough quantity cell pellets in order to proceed downstream work.

Key parameters to adjust:
A. Cell density (OD₆₀₀)
B. Transfer volumes from the 96 deep-well induction plates to 96-well unskirted plates

Follow proteomics sample preparation protocol:
Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format


2h 30m