Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre

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Latest revision as of 22:24, 27 October 2010

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)

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. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.

RESULTS

Raw growth curve

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

Raw GFP curve

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.

Tecan Test

@@ Line 28: / Line 28: @@
 *<partinfo>BBa_K173000</partinfo> (positive control)
 *<partinfo>BBa_B0031</partinfo> (negative control)
-They were let grow ON at +37°C, 220 rpm.
+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 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.
+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.
-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.
+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. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.
 ==RESULTS==