GENERAL INTRODUCTION

While we were at the laboratory developing our project, we also participated to the 2018 Interlab Study.

CALIBRATION PROTOCOLS

1. Calibration 1 : OD600 Reference point - LUDOX protocol

We added 100 μL of LUDOX into wells A1, B1, C1, D1 and 100 μL of dd H2O into wells A2, B2, C2, D2. We measured absorbance at 600 nm of all samples. OD obtained the resultes below:

2. Calibration 2 : Particule Standard Curve – Microsphere Protocol

Preparation of the Microsphere stock solution

We vortexed vigorously for 30 seconds the tube labeled “Silica Beads” from the InterLab test kit. We pipeted 96 μL microspheres into a 1.5 mL eppendorf tube and added 904 μL of dd H2O to the microspheres. We vortexed a second time and our Microsphere stock solution is ready to use.

Preparation of the serial dilutions of Microspheres

We added 100 µL of dd H2O into wells as below:

After, we vortexed vigorously the Microsphere stock solution and put 200 µL of it into well A1. We transferred 100 µL from A1 into A2, pipeted up and down few times and transferred 100 µL from A2 into A3. We continued this pipeting until reaching well A11. At the moment, we took 100 µL of A11 and put it on trash. We redid this protocol for rows B, C and D, as we will get replicates.

Thus, we made serial dilutions of our Microsphere Stock solution from well A, B, C and D1 to well A, B, C and D11 and A, B, C and D12 will be our blanks.

We get the results below:

3. Calibration 3: Fluorescence standard curve – Fluorescein protocol

Preparation of the fluorescein stock solution

We span down fluorescein kit tube from the InterLab test kit. We prepared a 10X fluorescein stock solution by adding 1 mL of PBS 1X to fluorescein tube et resuspending it. After, we prepared a 10 dilution of this stock to achieve a 1X fluorescein stock solution ready to use.

Preparation of the serial dilutions of fluorescein

We added 100 µL of 1X PBS into wells as below:

Secondly, we put 200 µL of our 1X fluorescein stock solution into well A1. We transferred 100 µL from A1 into A2, pipeted up and down few times and transferred 100 µL from A2 into A3. We continued this pipeting until reaching well A11. At the moment, we took 100 µL of A11 and put it on trash. We redid this protocol for rows B, C and D, as we will get replicates.

Thus, we made serial dilutions of our 1X fluorescein stock solution from well A, B, C and D1 to well A, B, C and D11 and A, B, C and D12 will be our blanks.

We get the results below:

Cell measurement protocol

We have at our disposal following devices from iGEM Distribution Kit, all in pSB1C3 backbone and from Kit Plate 7:

We resuspended each device in 10 µL of dd H2O and put them in tubes kept in our fridge.

1. Day 1 : Tranformation of these devices

We used an heat choc protocol for this transformation. We put 50 µL of DH5α competent cells in each tube and we added 2 µL of plasmid DNA. We let it cool 30 min on ice and made a 50 sec heat shock (at 42°C). We added 500 µL of LB and let grow 1h at 37°C at 185 rpm. Each tube were plated in one petri dish (LB + Chloramphenicol) and we incubated them overnight at 37°C.

2. Day 2 : results and cultures

Results of transformation : There are colonies on each plates. It will be possible to take 2 colonies to carry on with the interlab study. We made precultures of two clones per device named a and b with 2 mL of LB + Cm and let it grow overnight at 37°C at 185 rpm. We made overnight cultures from these precultures taking 150 µL of it and adding 5 mL of LB + Cm and keeping the rest in the fridge as a backup.

3. Day 3 : Cellule growth, sampling, and assay

After a 1 : 10 dilution of our cultures, we obtained following OD :

With those OD, we were able to do cultures at OD = 0,02 in a final volume of 12 mL LB + Cm. We took 500 µL of these samples placed into 1,5 mL Eppendorf tubes kept on ice during cultures’ growth during 6h at 37°C and 185 rpm. When we stopped growth, we kept 500 µL of growing cultures and we made measurements on both samples (T = 0h and T = 6h). Our layout for Abs600 and fluorescence measurement is the following one :