Inter Lab

What is InterLab?

InterLab Study is a measurement of GFP fluorescence among different lab organized by the iGEM community, aiming to produce common, comparable units for measuring GFP with different plate readers.

Our Protocol

Transformation: please refer to the protocol provided by iGEM community.
Calibration and Cell measurement: We are following the protocol provided by iGEM community.
Plate reader：BMG LABTECH: CLARIOSTAR MONOCHROMATOR-BASED MICROPLATE READER WITH COMPUTER & LASER PRINTER.

Calibration 1: OD600 Reference point - LUDOX Protocol

Calibration 2: Particle Standard Curve - Microsphere Protocol

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Cell measurement protocol

Day 1: Transform Escherichia coli DH5α with these following plasmids (all in pSB1C3):

Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

Day 3: Cell growth, sampling, and assay．At the end of sampling ，we measure the samples (Abs600 and fluorescence measurement) at two time point.

Fluorescence Raw Readings:

Abs600 Raw Readings:

Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

For the CFU protocol, we will need to count colonies for two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

Step 1: Starting Sample Preparation

Measure the OD600 of the cell cultures, making sure to dilute to the linear detection range of the plate reader,e.g. to 0.05 – 0.5 OD600 range. Include blank media (LB + Cam) as well.

Step 2: Dilution Series Instructions

There are 12 total Starting Samples prepared in step 1 - 6 for Positive Controls and 6 for Negative Controls.
For each Starting Sample (total for all 12 showed in italics in paraenthesis): dilute each starting sample until dilution 5 , and aseptically spread plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5.
Incubate at 37°C overnight and count colonies after 18-20 hours of growth.

Step 3: CFU/mL/OD Calculation Instructions

Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows: