Part:BBa_K300002:Experience
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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, 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 with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. 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_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 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 metabolic burden due to the synthesis of the studied fusion proteins. It is possible to see that all doubling times are comparable; it is possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to the cells.
From GFP curve it is possible to appreciate that in BBa_K300086, BBa_K300088, BBa_K300090, BBa_K300099 GFP accumulation is very similar and it is significantly different from the one of the 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 exponential growth phase, showing again an appreciable GFP production rate that is about half of the positive control GFP.
User Reviews
UNIQ8624dd72f37efbed-partinfo-00000014-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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UNIQ8624dd72f37efbed-partinfo-0000001B-QINU