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 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.
Fluorescence standard curve
We generated the fluorescein fluorescence standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence using the graph.
GFP expression in culture
We measured the OD600 values and fluorescence signal of the growing culture in fixed time interval. We processed the data by averaging the obtained values of two culture of different colonies with the same device. The results were represented in below graph:
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 (device 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The different in fluorescence per OD600 is probably due to the strength of promoter. Device 1 has the strongest promoter(J23101), thus it has a highest GFP production rate and give the highest fluorescence. Device 3 has a weakest promoter(J23117) , thus it has a lowest GFP production rate and give 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 (device 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 is a weaker RBS than B0034.
Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hours). These may because the increase in cell number is much faster than the overall expression of GFP after t= 2hrs.
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.
