Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre

SEPTEMBER, 28TH

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 and efficiency of inducible system assembled upstream of some genetic circuits.

Methods

Inoculum (into 5 ml LB+Amp) from glycerol stock of:

• I63
• I64
• I65
• <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. I63 and I64 circuits were also induced 100nM with HSL directly into multiplate well. 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 except for induced cultures. In this case doubling time is much higher than posite control and uninduced cultures; so it's possible to assert that in this case there's a kind of metabolic burden higher than in the others, maybe because of the inducible system.

In GFP curve it's possible to appreciate that in induced I63, I64 and I65 GFP accumulation profile it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. On the other hand not induced I63 and I64 show a profile very similar to the last one. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit and that the inducible system works as expected.

A mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate for I65 and a lower one for inducible constructs. Also not induced I64 and I65 show a low GFP rate synthesis maybe due to 3OC6HSL inducible circuit leakage activity.

Tecan Test