Interlab

Comparable Data Under Different Conditions

Interlab
Introduction
Protocol
Measurements
Provenance
Reference

Ⅰ.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 Device: I20270 in pSB1C3;
Negative Control Device: R0040 in pSB1C3;
Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3;
Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3;
Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3;

Figure 1. GFP generator with different promoters and R0040 in pSB1C3.
1. Positive Control Device in DH5α;
2. Negative Control Device in DH5α;
3. Test Device 1 in DH5α;
4. Test Device 2 in DH5α;
5. Test Device 3 in DH5α.

Digestion Verification

Figure 2. Restriction map of digestion verification. 2000plus DNA marker.
1. Double digestion of Test Device 1;
2. Double digestion of Test Device 2;
3. Double digestion of Test Device 3;
4. Double digestion of Positive Control Device.

Positive Control Device and three Test Devices are double digested at EcoR I and Pst I restriction sites to verify the target parts. The restriction map is shown as Figure 2. As expected, the second bands are supposed to be aroud 1000bp, which is consistent with what we saw on the map.

Ⅱ. Protocol

Cell measurement protocol

1. Transform five 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+ 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, 1, 2, 3, 4, 5 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, 520 nm emission.

Ⅲ. Measurements

1. OD600 Reference point

Table 1. Absorbance at 600nm for LUDOX and H2O

2. FITC standard curve

Table 2. Fluorescence for FITC at decreasing concentration

Figure 3. The FITC Standard Curve in plate reader with 485nm excitation, 520nm emission

3. Normalisation

Table 3. Absorbance at 600nm for five devices after cell growth for 16 hours and media+chl

4. Cell measurement

1. Abs600

Table 4. Absorbance at 600nm for five devices after blank substraction and correction

Figure 4. The plot of Abs600 versus time

2. Fluorescence

Table 5. Fluorescence for five devices after blank substraction

Figure 5. The plot of FI versus time in microplate reader with 485nm excitation, 520nm emission

3. Fl/Abs600

Table 6. The FI/Abs600 of five decives

Figure 6. The chart of FI/Abs600 versus time for five devices

4. Average and SD

Table 7. The average and SD of fluorescence for five devices

Ⅳ. Provenance

Q: Who did the actual work to acquire these measurements?
A: Zeyue Gao.

Q: What other people should be credited for these measurements?
A: Kainan Chen.

Q: On what dates were the protocols run and the measurements taken?
A: Required devices were transformed on 14th August, 2016. All samples were measured on 15th August, 2016.

Ⅴ.Reference

1. Anderson,C., Berkley iGEM Team., (2006). Anderson Promoter Collection. Registry of Standard Biological Parts.
2. http://parts.igem.org/Promoters/Catalog/Anderson [Accessed 2014.09.19]
3. https://2015.igem.org/Team:Amoy/Interlab