Overview

How do standard promoters behave in Mountain View, California, USA? We participated in the 2nd Annual InterLab Measurement study to contribute our results to a body of data from teams from around the world!

See our BioBricks

Background

The purpose of the 2015 InterLab Study is to measure the fluorescence of three devices: a high-, medium-, and low-strength promoters fused to a green fluorescent protein (GFP) generator. iGEM teams from around the world construct the same devices and compare results.

The three devices are:

Device 1: J23101 + I13504, in pSB1C3

Device 2: J23106 + I13504, in pSB1C3

Device 3: J23117 + I13504, in pSB1C3

For our positive control, we used the one suggested: GFP device BBa_I20270. For our negative control, we also used the one suggested: TetR repressible promoter.

All devices and parts were obtained from the 2015 Distribution Kit. Please see the link to our lab notebook below for more detailed information on device assembly. The results were confirmed with DNA sequencing by Elim Bio (see figure above).

We also participated in the Extra Credit Opportunity.

Data and Results

All results were measured in relative fluorescent units (RFU).
To standardize fluorescent measurements, the RFU measurements of each well were divided by the exact OD600 measurement of that same well.

Protocols

The chassis we used for the InterLab Study was E. coli NEB5-alpha, which is Biosafety Level 1. The personal protective equipment used included gloves and long pants. We used BioBricks assembly to construct all of the devices, and verified the sequences with sequencing done by Elim Bio.

To grow up the cells, we completed the following steps as specified in the InterLab Study Protocol Form:

1. Streak out one plate of LB agar with appropriate antibiotic per device and control.
2. Incubate plates for 17 hours at 37C (New Brunswick INNOVA 4200 Shaker Incubator).
3. Inoculate three biological replicates of 4mL liquid cultures in LB + chloramphenicol 15mL conical tubes.
4. Incubate liquid cultures for 18 hours at 37C, shaking at 190RMP and placed at an angle.
5. Samples were placed in 4C fridge for 24 hours before testing.
6. Samples were diluted 1:20 in LB and measured on a flow cytometer.
7. Use a plate reader and 96-well plate to perform OD600 and fluorescence measurements, using 200uL of samples and LB + chloramphenicol blanks.
8. Set instrument to read OD600, take measurements, calculate the dilution required to reach an OD600 of .5 ± 5%, perform the dilutions, and re-measure. Repeat this step until the OD600s are all .5 ± 5%.
9. Place 96-well plate with samples into fluorometer and measure with excitation at 485nm, emission 528nm, cut-off 495nm.
10. Plate reader data was processed using Microsoft Excel. Overall mean and standard deviation values were calculated across biological and technical replicates.