Team:SKLMT-China/InterLab
Introduction
Over the past four years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements.
This study aim to verify that whether CFUs could take the pace of OD during the exploration about green fluorescence protein’s production and cell growth in E. coli. DH5α cells. If measurements that are taken in different labs are no more variable than measurements taken within the same lab, this study will improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole.
It is a valuable experiment for the development of synthetic biology. So, we decide to participate in this meaningful study.
Results
1. Transformation of 8 test devices into competent E. coli.DH5α.
The transformation was successful for all the plasmids and resulted in a proper amount of colonies on all plates. After that, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol overnight (approximately 16-18 hours) at 37°C and 220 rpm.
Information about the devices:
| Devices | Part Number |
|---|---|
| Positive Control | BBa_I20270 |
| Negative Control | BBa_R0040 |
| Test Device 1 | BBa_J364000 |
| Test Device 2 | BBa_J364001 |
| Test Device 3 | BBa_J364002 |
| Test Device 4 | BBa_J364007 |
| Test Device 5 | BBa_J364008 |
| Test Device 6 | BBa_J364009 |
2. Measurement of LUDOX CL-X OD600 Reference Point.
LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a ratiometric conversion factor which allows us to transform absorbance (Abs600) data into a comparable OD 600 measurement.
It’s necessary for this study. Because, generally, plate readers do not have a 1 cm path length which means that measurements are volume dependent. However, to be convictive, measurements must be transformed to be standard.
By doing so, we obtained following data.
| Measurement | LUDOX CL-X | H2O |
|---|---|---|
| Replicate 1 | 0.057 | 0.037 |
| Replicate 2 | 0.057 | 0.036 |
| Replicate 3 | 0.057 | 0.036 |
| Replicate 4 | 0.058 | 0.036 |
| Arith. Mean | 0.057 | 0.036 |
| Corrected Abs600 | 0.021 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.000 |
3. Graphing a Particle Standard Curve.
With the Microsphere Stock Solution and the serial dilution of Microspheres prepared, particle standard curve was measured.
4. Graphing a Fluorescence Standard Curve
To compare fluorescence output of test devices between teams, a standard curve for fluorescence of Fluorescein was measured. This stand curve is used to correct our cell-based readings to an equivalent Fluorescein concentration.
Following the instructions, we obtained following results.
5. Measurement of OD600 and Fluorescence of Transformed Cells
With Abs600=0.02 at the begining, colonies were incubated 37 ℃, 220rpm. Then they were measured on plate reader at 0 hour and 6 hour.
6. Measurement of Colony Forming Units per 0.1 OD600 E. coli cultures
Finished “Starting Sample Preparation”, we got the “Starting Sample” with a 0.1 OD600 measurement. Then following the instructions, we obtained a series of dilution, and we counted the CFUs of dilution 3, dilution4, and dilution5 of positive control and negative control on plates.
Colony Forming Units per 0.1 OD600 E. coli cultures:
Discussion
In figure 1.5-2, the highest fluorescence date is measured from device 4, much stronger than positive control. Device 2 and positive control show a modest fluorescence intensity and device 5,6,1 show low fluorescence intensity, while test devices 3 have close fluorescence signal to negative control group.
Well, these devise have different data in fluorescence intensity. However, when CFUs of them are concerned about, convictive conclusion is not available temporarily. Several factors, like the moisture capacity of plates, methods in spreading plate, and time spent in each simple ect. result in different CFUs dates.
Conclusion
According to measurements above, 8 test devices all works in E. coli. DH5α cells, but different characters were shown in this interlab study.The OD results tells us that cells with device 4 and cells with device 5 grow happier. As for the fluorescence intensity, device 4 is the strongest member and device 3 is weaker than others.
Inter lab study is successfully finished, which gives us a hand in our experiment design and means a lot for the development of synthetic biology.