Interlab Study Project

Devices Measured

1. The first device is [http://parts.igem.org/Part:BBa_I20260 BBa_I20260], which is a biobrick part that contains:

A promoter, [http://parts.igem.org/Part:BBa_J23101 BBa_J23101], a downstream gene ([http://parts.igem.org/Part:BBa_E0240 BBa_E0240]), and a backbone ([http://parts.igem.org/Part:pSB1C3 pSB1C3]).

The downstream gene is also a composite of a number of biobrick parts, including an RBS - [http://parts.igem.org/Part:BBa_B0032 B0032], a GFP coding sequence - [http://parts.igem.org/Part:BBa_E0040 E0040], and a transcriptional terminator - [http://parts.igem.org/Part:BBa_B0015 B0015].

2. The second device was cloned in lab, as described in the interlab study procedure.

The device is composed of [http://parts.igem.org/Part:BBa_J23101 BBa_J23101] and [http://parts.igem.org/Part:BBa_E0240 BBa_E0240], which are the same parts as in the first. However, the backbone of this device is different, [http://parts.igem.org/Part:pSB3K3 pSB3K3]

3. The third device was also cloned in lab, as described in the interlab study procedure.

The device is composed of [http://parts.igem.org/Part:BBa_J23115 BBa_J23115] and [http://parts.igem.org/Part:BBa_E0240 BBa_E0240], which is the same downstream gene as in devices 1 and 2. However, the promoter sequence of device 3 is different. This device is in [http://parts.igem.org/Part:pSB1C3 pSB1C3].

Protocols

Sample Preparation

• Inoculate 10 mL of LB with frozen stocks of the three transformed clones (I20260, J23101+E0240, J23115+E0240), a cell background control (TOP10 electrocompetent cells), and an LB only control in a 50 mL Erlenmeyer flask. Grow for 16-18 hours at 37°C, shaking at 300rpm.
• The next morning, add 10µL of each overnight culture to 10 mL of LB with three replicates of each (15 total flasks) and grow 16-18 hours at 37°C, shaking at 300rpm.
• Add 80 µL of each culture to the wells of a clear-bottomed black 96-well plate.

Measurement Protocol

The samples were plated on a 96-well plate as follows:

• The 96-well plate was inserted into the Infinite 200 PRO Microplate Reader
• Open Tecan i-control, the software used to document the readings
• Configure the settings as detailed above
• Obtain fluorescence and OD600 readings

Sequencing Data