Team:XMU-China/Safety

2017.igem.org/Team:XMU-China/Safety

Safety
Introduction

The goal of the interlab study is to explore a major question: How close can the numbers be when fluorescence is measured all around the world? So we measure GFP fluorescence in our lab with plate reader (Tecan Infinite M200pro). This year, three devices and one positive control and one negative control were provided by the Registry. The results show increased fluorescence in the stronger promoter expected.


After the experiment, five required devices were created:
Positive control: I20270 in pSB1C3;
Negative control: R0040 in pSB1C3;
Test Device 1: J23101.BCD2.E0040.B0015 in pSB1C3;
Test Device 2: J23106.BCD2.E0040.B0015 in pSB1C3;
Test Device 3: J23117.BCD2.E0040.B0015 in pSB1C3;
Test Device 4: J23101+I13504 in pSB1C3;
Test Device 5: J23106+I13504 in pSB1C3;
Test Device 6: J23117+I13504 in pSB1C3.

Protocol

I. Materials

• Competent cells ( Escherichia coli strain DH5α)
• LB (Luria Bertani) media
• Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
• 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
• Incubator at 37°C
• 1.5 ml eppendorf tubes for sample storage
• Ice bucket with ice
• Pipettes

II. Cell measurement protocol

1.Transform 8 plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2.Pick 2 colonies from each of plate and inoculate it on 10mL LB medium and Chloramphenicol. Grow the cells for 16hrs at 37°C and 220rpm.
3.Cell growth, sampling, and assay.
4.Obtain initial OD 600 measurement of overnight cultures. Then dilute each sample to an OD of 0.02.
5.Incubate the cultures at 37°C and 220rpm. Take 100µL (1% of total volume) samples of the cultures at 0, 2, 4 and 6 hours of incubation.
6.At the end of sampling point measure these samples (OD and Fl measurement).
7.Measurements of absorbance and fluorescence:
(1) OD 600
Device: Plate Reader（Tecan Infinite M200pro） Wavelengths: 600 nm absorption.
(2) Fluorescence
Device: Plate Reader（Tecan Infinite M200pro）, 96-well plates. Wavelengths: 485 nm excitation, 530 nm emission.

Measurements

I. OD 600 Reference point


Table 1. Absorbance at 600nm for LUDOX and H2O

II. Fluorescein standard curve


Table 2. Fluorescence measurements for fluorescein stock solution at decreasing concentration


Figure 3. The Fluorescein Standard Curve in plate reader with 485nm excitation, 530nm emission