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At this moment, we started the characterization by inducing one of the flasks with IPTG to a final concentration of 20 mM. The other culture was not induced, as it was the negative control. Each flask was divided in 8 different tubes (one per hour) and incubated at the same conditions. When we performed a 10-hour test try, we determined that hour 8 resulted in the highest expression of RFP, so we limited the assay to 8 hours. Each hour, we took the corresponding tubes and took 3 mL into two different 1.5 mL microtubes. We used 100 μL to measure absorbance at 600 nm using the multiwell plate and 100 μL to measure fluorescence using the Synergy H1 Hybrid Multi-Mode Reader of BioTek. Program the excitation and emission wavelength at 550 nm and 584 nm respectively. The tubes were centrifuged at 5000 rpm for 2 minutes and we discarded the supernatant. The pellets were preserved at -20ºC. As we can see in the figure 1, the OD of the IPTG induced sample was less than the non-induced sample, nevertheless, the RFP was more expressed in the induced samples.</br></br> | At this moment, we started the characterization by inducing one of the flasks with IPTG to a final concentration of 20 mM. The other culture was not induced, as it was the negative control. Each flask was divided in 8 different tubes (one per hour) and incubated at the same conditions. When we performed a 10-hour test try, we determined that hour 8 resulted in the highest expression of RFP, so we limited the assay to 8 hours. Each hour, we took the corresponding tubes and took 3 mL into two different 1.5 mL microtubes. We used 100 μL to measure absorbance at 600 nm using the multiwell plate and 100 μL to measure fluorescence using the Synergy H1 Hybrid Multi-Mode Reader of BioTek. Program the excitation and emission wavelength at 550 nm and 584 nm respectively. The tubes were centrifuged at 5000 rpm for 2 minutes and we discarded the supernatant. The pellets were preserved at -20ºC. As we can see in the figure 1, the OD of the IPTG induced sample was less than the non-induced sample, nevertheless, the RFP was more expressed in the induced samples.</br></br> | ||
We performed also two different 10% SDS PAGE for visualizing the 26 kDa band corresponding to the red fluorescent protein. Each gel was loaded with the non-induced sample and its induced counterpart. However, during the previous treatment, we did not incubate the samples at 100ºC in the water bath, while we did for the samples of the other gel. By not performing this step, the protein was not denaturalized and was seen under the transilluminator as figure 3 shows. In this figure we can also see that by each hour the expression of this protein increases until hour 8. </br></br> | We performed also two different 10% SDS PAGE for visualizing the 26 kDa band corresponding to the red fluorescent protein. Each gel was loaded with the non-induced sample and its induced counterpart. However, during the previous treatment, we did not incubate the samples at 100ºC in the water bath, while we did for the samples of the other gel. By not performing this step, the protein was not denaturalized and was seen under the transilluminator as figure 3 shows. In this figure we can also see that by each hour the expression of this protein increases until hour 8. </br></br> | ||
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Revision as of 08:25, 31 October 2017
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Characterization of BBa_J04450
The objective of this characterization was to evaluated the moment of maximum expression of the protein and its behavior with the induction with IPTG.
IPTG Curve of DH5a
The samples were taken four times and the values that show the tables are the averages. Table 1. RFP characterization absorbances for both IPTG-induced and non-induced samples of DH5a
IPTG induced | Non-induced IPTG | ||
---|---|---|---|
Hour | Abs at 600 nm | Hour | Abs at 600 nm |
0 | 0.52 | 0 | 0.52 |
1 | 0.4285 | 1 | 0.46275 |
2 | 0.48675 | 2 | 0.49125 |
3 | 0.57875 | 3 | 0.6225 |
4 | 0.621 | 4 | 0.63875 |
5 | 0.701 | 5 | 0.72175 |
6 | 0.749 | 6 | 0.761 |
7 | 0.7395 | 7 | 0.8155 |
8 | 0.862 | 0.86325 |
IPTG induced | Non-induced IPTG | ||
---|---|---|---|
Hour | Ex at 550 nm Em at 584 nm | Hour | Ex at 550 nm Em at 584 nm |
0 | 15349.75 | 0 | 17408 |
1 | 17248.25 | 1 | 13648.25 |
2 | 18731.75 | 2 | 13956.25 |
3 | 22942.5 | 3 | 16434.75 |
4 | 32991.75 | 4 | 15492.5 |
5 | 36092.75 | 5 | 20840.25 |
6 | 35503 | 6 | 23045 |
7 | 52331.75 | 7 | 26362 |
8 | 59337 | 32690 |