Automated combinatorial media preparation

Destination plate:

Water plate:

Source plate (with stock components):

Liquid handler:

Equipment

Value	Label
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:

• Kanamycin

To be prepared fresh for every cycle:

• 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

Input:

• total volume of the culture (we use 1500µL as total volume)
• standard media recipe (e.g. standard_recipe_concentrations.csv)

Run the notebook A_Find_Stock_Concentrations.ipynb.

Use the list of components concentrations created in step 5 and prepare stock solutions. Ideally, you should prepare enough volume for all DBTL cycles.

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 24-well_stock_plate_high.csv 24-well_stock_plate_low.csv 24-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. 1mL of the 300x concentration in the stock plate, we need a 3.33 dilution, so 300µL of Kan and 1000-300=700µL of H2O

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: target_concentrations.csv

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 dest_volumes.csv
• source plate instructions 24-well_stock_plate_high.csv
• biomek files P20_components.csv P20_culture.csv P20_kan.csv P20_water.csv P200_components.csv P200_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.

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.

Prepare the plate with fresh stocks:

FeSO4 :

• To prepare 20mL of 60millimolar (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 20mL 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 0.3millimolar (mM) using the formula: 9.e. 10mL * 0.3mM / 60mM = 0.05 mL of FeSO4

• Add 9.95mL of H2O to another (falcon) tube and 0.05mL 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 10000rcf
• 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

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:

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

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

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

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 30°C

Humidity 80%

Length of the run 48h 0m 0s

Shake Speed (800 rpm)

Filters:

• ID401 (620nm for biomass measurement)
• ID402 (pH measurement)
• ID403 (DO measurement)

On Multitron:

Temperature 30°C

Shake speed 700RPM

Place plate in Infors Multitron

Follow a protocol for absorbance assay from an m2p plate.

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.