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Template:UNIPV-Pavia/2010/HSLtag
From 2010.igem.org
Methods
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
- <partinfo>BBa_K300091</partinfo>
- <partinfo>BBa_K300092</partinfo>
- <partinfo>BBa_K173000</partinfo> (positive control)
- <partinfo>BBa_B0031</partinfo> (negative control)
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 cell 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-hours' experiment with measurements of absorbance and green fluorescence every five minutes using TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> circuits were induced 100nM with HSL directly into multiplate well. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.
Results
 |
 |
 |
| Culture | Doubling time [min.] ± std error |
|---|---|
| <partinfo>BBa_K173000</partinfo> | 76.3336 ± 1.4362 |
| <partinfo>BBa_K300091</partinfo> induced | 121.1434 ± 7.0275 |
| <partinfo>BBa_K300091</partinfo> not induced | 74.4267 ± 1.3696 |
| <partinfo>BBa_K300092</partinfo> induced | 122.6088 ± 1.2785 |
| <partinfo>BBa_K300092</partinfo> not induced | 71.5105 ± 2.7113 |
| <partinfo>BBa_B0031</partinfo> | 70.8421 ± 2.2181 |
Discussion
All cell cultures showed a similar growth curve; doubling time was computed as described here in order to have informations about the burden due to the synthesis of such fusion proteins. It's possible to see that all doubling time are very similar except for induced cultures. In this case doubling time is much higher than posite control and not induced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.
From GFP curve it's possible to appreciate that in induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a profile very similar to the last one. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded and that the inducible system works as expected.
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an GFP production rate that is different from negative control. Not induced <partinfo>BBa_K300091</partinfo> and <partinfo>BBa_K300092</partinfo> show a low GFP synthesis rate maybe due to 3OC6HSL inducible circuit leakage activity.
