Template:UNIPV-Pavia/2010/HSLtag
Methods
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
- BBa_K300091
- BBa_K300092
- BBa_K173000 (positive control, J23100 constitutive promoter expressing GFP)
- BBa_B0031 (negative control, a non-fluorescent culture)
Cultures were grown ON at 37°C, 220 rpm.
The following day cultures were diluted 1:100 and let grow again for about five hours at 37°C, 220 rpm.
The optical density (O.D.) of each culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0.02.
Then we performed a 21-hour experiment with measurements of absorbance and green fluorescence every five minutes using TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. BBa_K300091 and BBa_K300092 constructs were induced with 100nM of HSL directly in the 96-well microplate. Acquired data were blanked by subtracting the media absorbance (for absorbance measurements) and the BBa_B0031 fluorescence (for fluorescence measurements). Then, the relative GFP synthesis rate per cell was evaluated by computing (1/O.D.600)*dGFP/dt, where O.D.600 is the blanked absorbance of the culture of interest and GFP is its blanked fluorescence. Each value shown below is the mean of three measurements in exponential phase and error bars represent the 95% confidence interval of the mean.
Results
 Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - BBa_E0040) |
| Culture | Doubling time [min.] ± std error |
|---|---|
| BBa_K173000 | 76.3336 ± 1.4362 |
| BBa_K300091 induced | 121.1434 ± 7.0275 |
| BBa_K300091 not induced | 74.4267 ± 1.3696 |
| BBa_K300092 induced | 122.6088 ± 1.2785 |
| BBa_K300092 not induced | 71.5105 ± 2.7113 |
| BBa_B0031 | 70.8421 ± 2.2181 |
Discussion
All the cultures showed a similar growth curve; doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to obtain information about the burden due to the synthesis of such fusion proteins. It is possible to see that all doubling times are very similar except for induced cultures. In this case doubling time is much higher than both positive control and non-induced cultures; for this reason it is possible to assert that induction gives a high metabolic burden.
From GFP curve and mean protein synthesis rate it is possible to appreciate that induced BBa_K300091 and BBa_K300092 GFP accumulation profiles are comparable and they significantly differ from the GFP raw time series of the negative control BBa_B0031. On the other hand not induced BBa_K300091 and BBa_K300092 show a profile that is very similar to the negative control. These results show that the green fluorescent protein assembled downstream of the construct is correctly folded and that the inducible system works as expected.
Not induced BBa_K300091 and BBa_K300092 show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.