The purpose of this experiment is to induce protein expression by using isopropyl β-D-1-thiogalactopyranoside (IPTG) to bind to the lac repressor to inhibit it. Additionally, doing back dilutions of the bacteria ensure that the IPTG is added during the bacteria’s log phase for optimal protein production.
Step 1: Select 4 – 10 colonies from Agar plates and inoculate them in LB containing the correct antibiotic
Step 2: Using a sterile inoculation loop or pipette tip select a single colony from the LB-Agar plate
Step 3: Swirl inoculation loop or pipette tip containing colony in 5mL of LB containing the desired antibiotic. Incubate culture at 37ºC overnight (12-18 hours) shaking at 250rmp
Step 4: From overnight cultures set up 4 IPTG conditions for each culture: 25uL, 5uL, 2.5uL, and 1.25uL
| Condition 1 (5uL IPTG) | Condition 2 (2.5uL of IPTG) | Condition 3 (1.25uL of IPTG) | Condition 4 (25uL of IPTG) |
|---|---|---|---|
| 5mL LB Media | 5mL LB Media | 5mL LB Media | 5mL LB Media |
| 5uL Cam25 | 5uL Cam25 | 5uL Cam25 | 5uL Cam25 |
| 5uL Kan30 | 5uL Kan30 | 5uL Kan30 | 5uL Kan30 |
| 100uL of Respective colony | 100uL of Respective colony | 100uL of Respective colony | 100uL of Respective colony |
Step 5: Incubate Conditions overnight at 37ºC shaking at 250rpm
A quick experiment to test if production of our compounds causes a detriment to the cell health.
Step 1: Set up a series of 10-fold dilutions using a 96 well plate by adding 90 uL of LB into each well for each testing condition. Create three 10-fold dilution options and leave a blank well in each column. Each row will represent a dilution, and each column will represent a testing condition.
Step 2: Load 100 ul of each sample into the blank well in each column.
Step 3: Using a multichannel pipet, transfer 10ul of each well into the next well of 90ul of LB for the 10-fold dilutions. This will result in a 10-1, 10-2 and 10-3 fold dilutions
Step 4: Plate 2-5 uL of each dilution from the same clone onto a pre dried LB Agar plate. This will result in 4 LB plates, each with four rows of four.
Step 5: Incubate the plates overnight at 37C, image in the morning.