Team:Jiangnan China/InterLab
Overview
In the last 4 years, the Measurement Committee has been developing a robust measurement procedure for green fluorescent protein (GFP) though the Interlab study, aiming to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. And this year, we are delighted to join the study to know if we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.
Materials
96-spot well plates: clear plates from Corning
Bacteria strains: E.coli DH5α
Test devices: obtained from the distribution kit
| Device | Part number | Location |
|---|---|---|
| Position control | BBa_R0040 | Well 2D |
| Negative control | BBa_I20270 | Well 2B |
| Test Device 1 | BBa_J364000 | Well 2F |
| Test Device 2 | BBa_J364001 | Well 2H |
| Test Device 3 | BBa_J364002 | Well 2J |
| Test Device 4 | BBa_J364007 | Well 2L |
| Test Device 5 | BBa_J364008 | Well 2N |
| Test Device 6 | BBa_J364009 | Well 2P |
Results
Particle Standard Curve
Particle Standard Curve (log scale)
Fluorescein Standard Curve
Fluorescein Standard Curve (log scale)
uM Fluorescein / OD
Net Fluorescein a.u.
Net Abs 600
MEFL / particle
Summery
We studied for a time before doing interlab, and spent one-two day preparing experimental materials, such as preparing E. coli DH5α and several culture media. After full preparation, we started the experiment. In the first experiment, our antibiotics failed, causing too much bacteria to colonize the plate.The second time we had a problem with the competent cells, so the bacteria did not grow after the plate was painted.The third time, we reprepared the competent cells and replaced the antibiotics, and the experiment went well.
Conclution
We studied for a time before doing interlab, and spent one-two day preparing experimental materials, such as preparing E. coli DH5α and several culture media. After full preparation, we started the experiment. In the first experiment, our antibiotics failed, causing too much bacteria to colonize the plate.The second time we had a problem with the competent cells, so the bacteria did not grow after the plate was painted.The third time, we reprepared the competent cells and replaced the antibiotics, and the experiment went well.
1. The final concentration of chloramphenicol is not clearly defined in the experimental protocol. Although we have made the experiment proceed smoothly based on experience, we believe that accurate antibiotic dosage requirements are still necessary;
2. Regarding the use of the plate reader: although there are some descriptions in the experimental protocol that explain the experimental design and a simple explanation of the plate reader, we used the plate reader incorrectly at the beginning of the experiment, leading to a invalid data. So we still expect more instructions on the setup and use of the plate reader;
3. Regarding the preservation of competent cells: we have transferred the competent cells position between buildings due to the change of the laboratory position, which took about 10 minutes. And in the transformation experiment, the competent cells stayed outside for too long. These improper storage of competent cells made the bacteria grow very unsatisfactorily after plating. Therefore, the preservation of competent cells is very important.
