5.2 Competition Assay (Overnight Cultures)

Protocol for Library Growth with LB Media

Introduction

This protocol outlines the steps to grow up your libraries and perform chemical genomics for sequence quantification.

Materials

• Library
• Culture tubes
• Eppitubes
• LB Liquid media
• 250 mL flasks
• PCR tubes
• Falcon tubes

Procedure

Competition Fitness Assay with Chemicals

Prep

1. Assign Alphanumeric Labels: Assign alphanumeric labels for each sample and print out a "cheat sheet."
2. Calculate Dilutions for IPTG:
   • Volume of IPTG to add = (Final Volume of Culture / 1000)
   • Note: The dilution factor for IPTG is 1000 (from 1M to 1mM).
3. Prep 250 mL Flasks: Prepare 250 mL flasks for T0 collections.
4. Label Eppitubes: Prep Eppitubes with labels for pellet collection.

Day 1

1. Inoculate Flask: In a 250 mL flask, add 35 mL LB. To achieve a starting OD of 0.02, use the following volumes based on the stock OD:
   • For E. coli with stock OD = 25: Use 28 uL
   • For E. cloacae with stock OD = 30: Use 23 uL
   • For K. pneumoniae with stock OD = 30: Use 23 uL
   • Equation: Volume to inoculate = (Desired OD x Culture Volume) / Stock OD
2. Incubate: Place the flask in a 37°C shaker until the OD reaches 0.2.
3. Collect T0 Samples: Take 10 mL from each flask and distribute into Falcon tubes for each of the 3 replicates, including a backup for each. Note: 10 mL at OD 0.2 is approximately equivalent to 1 mL at OD 2.
4. Prep for DNA Extraction: Utilize the entire pellet for DNA extraction.
5. Prep Overnight Tubes for T1: Inoculate another overnight tube with 1 mL from the Falcon tube to achieve a starting OD of 0.02.
6. Incubate Tubes: Place tubes in a tube rack and incubate in a 37°C shaker for 18 hours.

Day 2 and Beyond

1. Measure OD: After 18 hours of incubation, extract 100 uL from each T1 tube. Dilute this into 900 uL of LB for a 1:10 dilution and measure the OD.
2. Prep for DNA Extraction: Take 1 mL, spin down, and store for DNA extraction. Take an additional 1 mL as a backup.
3. Prep for Next Timepoint (Tn): Inoculate another overnight tube with the aim of reaching an OD of 0.02 in 4 mL. Use 40 uL from the previous overnight.
4. Incubate Tubes: Place tubes in a 37°C shaker for 18 hours.

Repeat steps 1-4 for each subsequent day until you are done with the experiment.