Team:TecCEM/Improve
Improve
Characterization of BBa_J04450
The objective of this characterization was to evaluate the moment of maximum expression of the protein and its behavior with the induction with IPTG.
IPTG Curve of E. coli strain DH5α
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 E. coli strain DH5α
| 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 | |
Figure 1. Graph of the biomass growth of the induced and non induced cultures of E. coli strain DH5α + BBa_J04450 Table 2. RFP characterization absorbances for both IPTG-induced and non-induced samples of E. coli strain DH5α
| 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 | |
Figure 2. Statistical analysis of the measures of RFP obtained through 8 hours of induction.
Figure 3. Comparison between RFP expression in E. coli strain DH5α induced and not induced with IPTG.
Figure 4. Basic steps for RFP characterization.
Escherichia coli DH5α was transformed with BBa_J04450 in pSB1C3 and spatulated on LB+CAM (25 ug/mL) plates. To prepare the first inoculum, we resuspended an isolated colony from the plate into 10 mL of liquid LB broth with chloramphenicol and left incubating at 37ºC overnight with 250 rpm agitation. We prepared 2 flasks with 110 ml of LB broth with chloramphenicol and inoculated them with the previous culture. The flasks were incubated at 37 °C and 250 RPM until the OD600 reached 0.6. 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, which were programmed for 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 expressed more in the induced samples. We also performed 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 could be 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.
