Team:NAU-CHINA/InterLab
InterLab
Measurement Study
All competing iGEM teams were encouraged to participate in interlab study which aims to assess how close measurements with a robust procedure can be when fluorescence is measured all around the world. Reliable and repeatable measurement is a key component to all engineering biology. However, the ability to repeat measurements in different labs has been difficult.
Antifungal peptide collection
Day 1: transform Escherichia coli DH5α with these following plasmids:
Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1 (BBa_J364000): well 20F
Test Device 2 (BBa_J364001): well 20H
Test Device 3 (BBa_J364002): well 20J
Test Device 4 (BBa_J364003): well 20L
Test Device 5 (BBa_J364004): well 20N
Test Device 6 (BBa_J364005): well 20P
Day 2: Pick 2 colonies from each plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 2-20 rpm.
Day 3: Cell growth, sampling, and assay.
Measurements must be made in a plate reader, as the plate reader with a 96 well format is ideal as it provides a convenient format for multiple measurements. The first objective is to obtain standard curves for fluorescence based on the sodium fluorescein reference material provided. The standard curve must be obtained under exactly the same instrument conditions that you will use when you do your cell based expression assays.
Result
| LUDOX-HS40 | H₂O | |
| Replicate 1 | 0.044 | 0 |
| Replicate 2 | 0.041 | 0 |
| Replicate 3 | 0.043 | 0 |
| Replicate 4 | 0.044 | 0 |
| Arith. Mean | 0.043 | 0 |
| Corrected Abs600 | 0.043 | |
| Reference OD600 | 0.0425 | |
| OD600/Abs600 | 0.9883721 |
Table 1 shows the data for OD600 measured by a plate reader for the H2O and LUDOX. The corrected Abs600 is calculated by subtracting the H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the Reference OD600 divided by Abs600.
Figure 1. A standard curve generated by measuring the fluorescence of serial dilutions of FITC stock (uM). There is a linear trend for increased fluorescence as [FITC] increases.
Figure 2. A、B The fluorescence of the six test devices and both controls transformed into E.coli and inoculated in LB broth was measured every two hours over a 6-hour period. C、 D The absorbance of the six test devices and both controls transformed into E.coli and inoculated in LB broth was measured every two hours over a 6-hour period. The overall trend shows an increase in absorbance for all samples over time.
Figure 3. The fluorescence readings for each test device or control for every 2 hours reading. It can be seen that fluorescence intensity increased sharply in some devices. Error bars indicate variations in the results.
Discussion
During our experimental process, we found that the given time in transformation protocol was rather shorter than that we really cultured. And the transformation efficiency was pretty lower than we expected, and we were not the only one who ran into this situation.
