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SEPTEMBER, 27TH
PLATE
EXPERIMENT DESCRIPTION
Motivation
This experiment was performed to check bacterial growth and GFP synthesis rate of the following constructs in order to verify right protein folding.
Methods
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
- I47
- I48
- I49
- <partinfo>BBa_K173000</partinfo> (positive control)
- <partinfo>BBa_B0031</partinfo> (negative control)
They were let grow 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.
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.
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.
RESULTS
| Culture | Doubling time [min.] ± std error |
| <partinfo>BBa_K173000</partinfo> | 76.3336 ± 1.4362 |
| I47 | 73.6685 ± 1.6245 |
| I48 | 74.8806 ± 2.7699 |
| I49 | 75.9433 ± 3.6808 |
| <partinfo>BBa_B0031</partinfo> | 70.8421 ± 2.2181 |
 Mean of (dGFP/dt)/O.D.600 (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>) 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 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 cells.
From GFP curve it's possible to appreciate that in I47, I48, I49 GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. This result 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.
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