Yeast growth protocol in a bench bioreactor (SOP ID: 0010)

SOP ID:

0010

Revision nr.:

0.0

Release date:

November 19, 2019

Last release date:

January 28, 2020

Author(s):

Ada Serena Marletta

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for growing yeast cells into a 200 ml bench bioreactor. Drafting of this Procedure is in Progress.

2. Background

This Standard Operating Procedure is intended for qualified personnel who was trained according to the ISO-9001 quality framework.

3. Prerequisites

Documents required:

MIAMI quality manual
SOP ID 0001

Material required:

Generic lab-, glass- and plastic-ware
yeast glycerol stocks
isotherm rack
orbital shaker
Bioreactor Solaris IO 200
Hamilton pH probe
SC medium
SC agar plates
MicroClave Connectors
Himalia sterile syringe 3 mL with non-return valve (10 ml, 5 ml, 3 ml)
Duran® Connection Cap System GL25 with PBT cap ports, 2, stainless steel ports, PTFE insert
Duran Bottles
Tubing clamps, Squeeze-Fix
Eutectic plates
0.22 μm autoclavable filters
Bunsen burner
Silicone pipes
Spectrophotometer
Cuvettes
Polypropylene glycol P 2000
pH standard solutions
Ethanol

4. Changes since last revision

No changes were made since last version

5. Role and responsibilities

Author (Au): Staff scientist properly trained to draft, review and disseminate standard operating procedures within the framework of ISO-9001

Approver (Ap): PI with the authority to approve, reject and withdraw standard operating procedures

MIAMI Quality Manager (QM): MIAMI authorized Quality Manager

Operator (Op): Staff scientist properly trained to execute the standard operating procedure

6. Monitoring Requirements

The present document can be reviewed at any time required by the author and the MIAMI Quality Manager.

7. Procedure

Step

Action

Responsibility

Yeast strain revival:

Take the yeast glycerol stock from the main -80°C freezer and put it into an isotherm rack to prevent thawing.
Scrape the glycerol stock with a sterile toothpick or bacteriological needle.
Streak yeast cells on the appropriate plate and incubate at 30°C.

Yeast pre-culture preparation:

Pick a single colony from an SC agar plate
Culture picked colony in 3mL of SC medium (with the appropriate Synthetic Drop-Out Medium Supplements, if necessary) at 30°C, 250 rpm, O/N.
Transfer the content of the tube to a 250 mL baﬄed shake ﬂask containing 50mL SC medium (with the appropriate Synthetic Drop-Out Medium Supplements)and grow O/N at 30 °C shaking at 250 rpm.

Bioreactor pH calibration:

Switch on the computer and the process control system (PCS), then open the bioreactor’s software
Plug the pH probe and calibrate it with the standard pH solutions by clicking on the calibration menu and indicating the pH of the used solutions.
Switch off both PCS and the computer.

Bioreactor vessel setup :

Prepare 200 ml of SC medium and fill the bioreactor vessel with 150 ml
Ensure that the o-ring seal is properly seated on the upper edge of the vessel
Mount the vessel headplate and close it gently with the three screws. Do not screw hard to avoid liquid spills from the bioreactor
Put the protective cap on the pH probe and install it in the headplate.
Take a new MicroClave Connector and screw it to the headplate. No sampling syringe should be connected to the MicroClave Connector in this step
Place tubing on inlet and outlet ports as follows (according to Figure 1):

#	PORT	TUBING
2	Manual air inlet	Place a short piece of silicone pipe connected to a small 0.22 μm autoclavable filter and add another short piece of silicone pipe
3	three-ways inlet	Place two long silicone pipes (for acid and base supply) and one short silicone pipe (for the inoculum)
5	air outlet	Place one long piece of silicone pipe
7	air inlet	Place one short piece of silicone pipe and connect it with a big autoclavable 0.22 μm filter (622/CH)

Clamp each silicone pipe with a silicone tubing clamp and cover the open ends of tubes with foil
Close three empty DURAN bottles with GL25 caps and connect each cap to a small 0.22 μm filter via a small piece of silicone pipe
Sterilize both the bioreactor vessel and the three empty DURAN bottles in autoclave at 120 °C for 20 minutes
Prepare 500 mL of 0.1 M NaOH and 500 mL of 0.1 M H3PO4 in sterile pyrex bottles
Under laminar flow, fill one of the autoclaved DURAN bottles with 200 mL of 0.1 M NaOH solution and the second DURAN bottle with 200 mL of 0.1 M H3PO4
Connect the two long silicone pipes for acid and base supply placed on the feeding inlets to the acid and base DURAN bottles and block them on the respective peristaltic pumps: P3 is set for acid and P4 is for the base.
Connect the air outlet silicon pipe to the third autoclaved DURAN bottle dedicated to waste collection, letting the pipe pass through a polystyrene box filled with eutectic plates. This pipe needs to be as much vertical as possible, in order to reduce the loss of volume inside the bioreactor’s vessel by evaporation
Connect the air inlet

Inoculum preparation:

Measure the OD600 of the yeast pre-culture grown at step 2
Calculate the volume of yeast pre-culture needed to have a starting OD600 = 0.1 in 150ml working volume
Under laminar flow, withdraw the calculated yeast pre-culture volume, transfer it into a sterile tube and add SC sterilized medium up to 10 ml final volume

Yeast Growth Conditions setting:

Switch on the PCS and the computer, and access to the process control LEONARDO software
Switch on the air compressor by moving the knob on “auto” and set the pressure to 2 bars
Remove the protective cap from the pH probe, plug in the respective cable
Connect the stirrer motor
Run the “checkup sequence” on the LEONARDO software
Wait until the software checks if everything is properly connected
Click on “proceed” and start a new workflow
Click on “Synoptics” and manually fill the acid and base pipes by clicking on the specific button, setting the pump to “manual” and press the green button until you see the whole pipe filled with liquid. During pipe filling, be sure that no drops of acid/base solutions are injected in the vessel, in order to maintain the desired pH.
Set the two pumps to “auto”
Set culture conditions as follows and start the workflow:

Temperature	30°C
pH	5
Agitation	250 rpm
Mass flow	240 Nml/min

Sampling for OD600 blank measurement:

Right before inoculation, take 10 ml of sterilized culture medium according to SOP ID:0012. 1ml will be used to set blank for optical density measurement at 600 nm, and the remaining volume to dilute samples (if necessary).

Inoculation and sampling:

Under laminar flow, take the inoculum volume prepared at step 5 with the help of a sterile 10 ml syringe with the needle on
Remove the needle from the syringe and switch on the Bunsen burner close to the three-ways inlet
Remove the foil from the feeding inlet and inoculate yeast.
Remove the syringe from the feeding inlet
On the monitor click on “inoculum”
Fill a new sterile 5 mL syringe with 5 mL of sterile SC medium and leave it connected to the feeding inlet used for yeast inoculum
Switch off the Bunsen burner
Take a sample, according to SOP ID:0012, at the following time points: 10 min., 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours.
Increase the speed of the stirrer by 100 rpm after each sample collection

Export .csv files and proceed to data analysis.

8. Record Management

When completed and approved, the SOP will be published on MIAMI website

9. References

9.1 Standards

ISO-9001:2015
International System of Units

9.2 Other SOPs

Sampling procedure for yeast culture (SOP ID: 0012)

9.3 Reagents

For reference reagents, please refer to the Prerequisites section.

9.4 Sequences

The execution of this SOP does not refer to any reference sequence

9.5 Supplementary Documents

There are no supplementary documents.

10. Definitions

Measurements units are defined according to International System of Units

Metric prefixes are defined according to Metric (SI) Prefixes

Whenever possible, biological construct are defined according to the SBOL standard

Whenever possible, biological experiments are defined according to the metrology standards

11. Disclaimers

This Standard Operating Procedure (SOP) was developed based on a compilation of best available information, knowledge, experience, and general industry practices to provide guidance to MIAMI staff in performing the activities defined herein, in a consistent and standardized manner. This document does not contain regulatory or statutory requirements unless specified. The MIAMI consortium has made every attempt to present the information in a clear and concise manner for a variety of users. However, the MIAMI consortium is not responsible for the misuse or misinterpretation of the information presented herein. Under no circumstances shall The MIAMI consortium be liable for any actions taken or omissions made by non-MIAMI users of this document. In general, this document should be used as a reference. Differences may exist between the procedures referenced in this document and what is appropriate under project-specific conditions. This document does not represent an endorsement of practitioners or products mentioned in the document.

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