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

SEPTEMBER, 27TH

PLATE

EXPERIMENT DESCRIPTION

Motivation

This experiment was performed to check bacterial growth and GFP rate synthesis 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

Raw growth curve

Raw GFP curve

Mean of (dGFP/dt)/O.D.600 (under the hypothesis that half-life of GFP is longer than experiment observation)

All cell cultures showed a similar growth curve and 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 stress to the cells.

In 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>.

After an initial interval in witch GFP production rate was significantly higher in positive control <partinfo>BBa_K173000</partinfo> than in the other samples, GFP synthesis rate of I47, I48, I49 became very similar to this one. It was still lower than positive control, but was constant during all the experiment. This showed the right folding of the green fluorescent protein assembled downstream of the genetic circuit.

Tecan Test