Difference between revisions of "Part:BBa K300002:Experience"
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*<partinfo>BBa_K173000</partinfo> (positive control) | *<partinfo>BBa_K173000</partinfo> (positive control) | ||
*<partinfo>BBa_B0031</partinfo> (negative 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 | + | 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 with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted. | ||
| − | |||
====Results==== | ====Results==== | ||
<div align="center"> | <div align="center"> | ||
| Line 60: | Line 61: | ||
====Discussion==== | ====Discussion==== | ||
| − | All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It | + | All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells. |
| − | + | From GFP curve it's possible to appreciate that in <partinfo>BBa_K300086</partinfo>, <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded. | |
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control. | The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control. | ||
Revision as of 19:50, 27 October 2010
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Applications of BBa_K300002
This part was tested through BBa_K300086.
Methods
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
- BBa_K300086
- BBa_K300088
- BBa_K300090
- BBa_K300099
- BBa_K173000 (positive control)
- BBa_B0031 (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 with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.
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_K300086 | 73.6685 ± 1.6245 |
| BBa_K300088 | 74.8806 ± 2.7699 |
| BBa_K300090 | 75.9433 ± 3.6808 |
| BBa_K300099 | 78.4634 ± 2.5622 |
| BBa_B0031 | 70.8421 ± 2.2181 |
Discussion
All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.
From GFP curve it's possible to appreciate that in BBa_K300086, BBa_K300088, BBa_K300090, BBa_K300099 GFP accumulation it's very similar and it's significantly different from that of negative control BBa_B0031. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.
User Reviews
UNIQbb655bd54a660a4f-partinfo-00000013-QINU
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UNIPV-Pavia iGEM 2010 |
This part was used as a head domain alone and to construct the following synthetic composite affinity tags:
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UNIQbb655bd54a660a4f-partinfo-0000001A-QINU