Microplate used: Corning® clear flat bottom black 96 well plates
Instrument used: BMG LABTECH’s CLARIOstar®
| Instrument Settings for measuring OD600 of LUDOX and Cells: |
| Endpoint settings |
| No. of flashes per well |
10 |
| Scan mode |
orbital averaging |
| Scan diameter [mm] |
3 |
| Optic settings |
| Excitation |
600 |
| General settings |
| Top optic used |
| Settling time [s] |
0.1 |
| Reading direction |
bidirectional, horizontal left to right, top to bottom |
| Target temperature [°C] |
25 |
| Instrument Settings for measuring fluorecence of Fluorescein and GFP: |
| Endpoint settings |
| No. of flashes per well |
8 |
| Scan mode |
orbital averaging |
| Scan diameter [mm] |
3 |
| Optic settings |
| Excitation: |
470-15 |
| Emission |
515-20 |
| Gain |
500 |
| Focal height [mm] |
9 |
| General settings |
| Top optic used |
| Reading direction |
bidirectional, horizontal left to right, top to bottom |
| Target temperature [°C]
| 25
|
Results
The colonies was observed from blue light box.
The controls show that the plasmid containing GFP gene will give fluorescence.
The strength of fluorescence is correlated to the strength of promoter. The higher the strength of promoters, the brighter the colonies. Among the colonies from devices 4-6, the colonies from device 4 show the highest fluorescence while those from device 6 show the lowest.
Among the colonies from devices 1-3, the colonies from device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies from devices 1 and 2.
Fig.1 shows an increasing trend of the cell growth under OD600 absorbance
Fig.2 shows an increasing trend of the fluorescence.
Fig.3 shows the trend of fluorescence per cell.
Among devices 4-6, the data match with the fluorescence of their colonies and the strength of their promoters. Device 4 has the highest fluorescence per cell and device 6 has the lowest. Among devices 1-3, the data also match with the fluorescence of their colonies and the strength of their promoters. Device 1 has the highest fluorescence per cell and device 3 has the lowest.
Interestingly, the fluorescence per cell of device 6 and negative control are very close. The fluorescence per cell of device 6 is even lower than the negative control at some time points.
This shows that device 6 produces very little amount of GFP and the result may also be affected by the measurement error in plate reader.
Another interesting point is the fluorescence per cell of all devices peaked at (t=2hours). One of the possible reasons is the cells spend more energy on cell division but not on producing GFP.
Reference
1. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60.
