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
GFP expression in colonies
The colonies were observed from blue light box.
The controls show that the plasmid containing GFP gene will give fluorescence.
The strength of fluorescence correlates to the strength of constitutive promoters. The higher the strength of promoters, the brighter the colonies. Among the colonies with devices 4-6, the colonies with device 4 showed the highest fluorescence while those with device 6 showed the lowest.
Among the colonies with devices 1-3, the colonies with device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies with devices 1 and 2.
Fluorescence standard curve
We generated the fluorescence-[fluorescein] standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence from the graphs.
GFP expression in culture
We measured the cell density (OD600 values) and fluorescence signal of the cell culture at 2-hour interval. We processed the data by averaging the obtained values from two flasks of culture of different colonies with the same device. The results were represented in the graph below:
The increasing trends of OD600 and fluorescence in all devices are shown in the first and the second graph.
Among the devices that share the RBS B0034 (devices 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The difference in fluorescence per OD600 is probably due to the promoter strength. Device 1 has the strongest promoter(J23101), thus it gives the highest fluorescence. Device 3 has the weakest promoter(J23117) , thus it gives the lowest fluorescence. The strength of promoter can also be deduced by comparing the fluorescence per OD600 given by the devices that share the BCD2 (devices 4, 5, 6). Device 4 has the strongest fluorescent signal because it uses the strongest promoter (J23101), while device 6 has a weakest fluorescent signal because the weakest promoter(J23117) is used.
Comparing the fluorescence signal given by devices with the same promoter but different RBS, we observed that the devices that use BCD2 (devices 4,5 and 6) gave a lower fluorescence comparing with the devices that use B0034 (devices 1, 2 and 3). We may therefore conclude that BCD2 has a weaker RBS than B0034.
Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hour). These may because the increase in cell number is much faster than the overall expression of GFP after t=2 hour.
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.
