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JUDGING FORM ⇗

2018 iGEM Interlab

Introduction

As the field of synthetic biology emerges as a viable solution to the world’s problems, it will experience many of the growing pains other fields have faced. One such challenge is how to communicate the methods and results of ones experiments effectively. The iGEM Interlab Study has worked for the past 5 years to correct this issue. Last summer, teams took part in the 4th iGEM Interlab Study. In which, teams across the planet contributed data in an effort to reduce variability in GFP measurements by normalizing for optical density. To build upon this, the 5th iGEM Interlab Study attempts to reduce error in fluorescence readings by normalizing measurements to colony forming units (CFU).

Methods: All procedures conducted can be found in full in the 2018 Interlab Plate Reader Protocol.

OD600 Reference Point - Ludox Protocol

Procedure

Assembling Gel Bed

1. Added 100 μl LUDOX into wells A1, B1, C1, D1
2. Added 100 μl of dH2O into wells A2, B2, C2, D2
3. Measured absorbance at 600 nm of all samples

Table 1. OD600 Reference Point

	LUDOX CL-X	H2O
Replicate 1	0.063	0.024
Replicate 2	0.058	0.023
Replicate 3	0.056	0.024
Replicate 4	0.058	0.025
Arith. Mean	0.059	0.024
Corrected Abs600	0.035
Reference OD600	0.063
OD600/Abs600	1.813

Base on our results, the OD600/Abs600 conversion factor for our spectrophotometer is 1.813.

Particle Standard Curve - Microsphere Protocol

Procedure

1. Vortex the tube labeled “Silica Bead” (obtained from the iGEM Interlab Kit)
2. Immediately pipetted 96 μL microspheres into a 1.5 mL eppendorf tube
3. Add 904 μL of dH2O to the microspheres
4. Vortexed and set aside as the new silica bead stock

Serial Dilution of Microspheres

Procedure

1. Added 100 μL of dH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
2. Vortexed the silica bead stock
3. Added 200 μL of the silica bead stock to A1
4. Transferred 100 μL from well A1 to well A2 and mix by pipetting
5. Repeated step 4 for wells A2 through A11
6. Discard 100 μL from A11
7. Repeated steps 2 through 6 for rows B through D

Note: Before being placed into the plate reader, all wells were mixed again by pipetting.

Fluorescence Standard Curve - Fluorescein Protocol

Procedure

1. Spun down the fluorescein kit tube (obtained from the iGEM Interlab Kit)
2. Prepared a 10x fluorescein stock solution by resuspending the fluorescein in 1 mL of 1x PBS
3. Created 1x fluorescein stock solution (10 μM) by diluting 100 μL of the 10x fluorescein stock with 900 μL of 1x PBS

Serial Dilution of Fluorescein

Procedure

1. Added 100 μL of 1x PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
2. Added 200 μL of 1x fluorescein stock solution into wells A2, B2, C2, D2, E2, F2, G2, H2
3. Transferred 100 μL from well A1 to well A2 and mix by pipetting
4. Repeated step 4 for wells A2 through A11
5. Discard 100 μL from A11
6. Repeated steps 2 through 6 for rows B through D

Note:The plate reader used was a Promega Glomax Multi Detection System and the filters used were all sterile and performed under aseptic conditions

Cell Measurement Protocol

Day 1: The following devices were transformed (link to transformation protocol) into DH5 - alpha competent cells.

Day 2: Two colonies were picked from each of the transformation plates and inoculated in 5 mL of LB + Chloramphenicol medium. And grown overnight for 18 hours at 35 degrees Celsius and 250 rpm.

Day 3:

1. Created 1:10 dilution of each overnight culture in LB + Chloramphenicol (0.5mL of culture into 4.5 mL of LB + Chloramphenicol
2. Measured Abs 600 of these 1:10 diluted cultures
3. Diluted the cultures to an Abs 600 of 0.02 12 ml LB + Chloramphenicol in 50 mL falcon tubes that were immediately covered in foil
4. 500 μL of each sample were transferred into individual 1.5 mL tubes as 0 hour samples prior to incubation and placed on ice
5. Incubated the remainder of the cultures for 6 hours at 37 degrees Celsius at 220 rpm
6. Took 500 μL of each sample and placed them on ice
7. Measured Abs 600 and fluorescence after adding 100 μL of the respective samples as shown in figure below

All raw data can be found on our excel spreadsheet: The Univeristy of Iowa iGEM 2018 INTERLAB RESULTS