Results

Description of the results work

Finally, the “Sound of Coli” project is completed even if with some difficulties. We have been able to analyse the interaction of the cytoplasmic oscillator using the multi-plate reader Tecan. The Tecan experiment was done the last 2 weeks of October. The first time we examined the reactions of our system in different conditions, both individually and together; when our two strains were inserted together in the same well we used different proportions 1:1, 10:1, 100:1 and unfortunately, because we had few inoculum, the dominant strain was always system 1 strain. The second time we tried the Tecan reader we started from a bigger cultivation so we were able to study the other proportions with the majority of system 2 strain too. Unluckily we were not able to analyse this second data experiment, but we are sure they are as good as the first data experiment.
So, from the first experiment we observed that it is stronger the tetracycline induction of the oscillator, because the red fluorescence of the system 2 strain has a constant growth.

As we can notice from this graphic, in every situation of different proportions of the two strains and of different condition of the inducers (tetracycline and IPTG) the GFP’s emission is oscillating during the time while the RFP’s emission has a kind of linear growth.

This kind of trend is still visible in the graphics of the 1:1 ratio of the two fluorescence.

Instead, by observing the 10:1 and 100:1 proportions it clearly appears the decreasing in the GFP/RFP ratio and the parallel increase of the RFP/GFP ratio. This data show that the two recombinant strains communicate and influence each other, even if the cycle of induction and inhibition favors the system 2 which generates the RFP.
If we focus on the grey and yellow lines, that stands for the induction conditions where is present tetracycline, we can point out that these lines in the GFP/RFP graph are the lower and they are the higher in the RFP/GFP plot. This is a correct info according to how the two systems are constructed because the RFP’s production is stimulated by tetracycline and the GFP’s is inhibited by the antibiotic.

Future plans

According to these results, our primary goal to improve this project should be experiment various concentrations of the inductors and different dilutions of the two strains to catch a more oscillating plot.
Another development of the project could be to change the first induction: now the oscillator is activated by two laboratory molecules tetracycline and IPTG but we think it is possible to create an induction mediated by a sensor. For example, a molecule can be captured by a receptor which stimulates a signal transduction that brings the system to induct the genic oscillator, that will emit a kind of fluorescence according to the external conditions. This plan was part of our project: we elaborated a third system, capable of communication with the others through the same quorum sensing molecules, in which the cyan fluorescence was activated by the presence of copper in the cultivation media. Since we had problems to assembly the genic sequence in the lab, we were forced to cut off this final part of the oscillator. Even if we failed this summer in this part, we still think it is valid upgrade of this project because the sensor oscillator could induce a metabolic pathway to resolve (positively or negatively) the message brought by the sensory molecule! It is also valid because this trick could transform a science art project in a valid method to measure some molecules.

Consideration for replicating the experiments

If someone would like to replicate our experiment, or maybe to do new implements in the project too, we suggest: to work on DH5α strain of E. coli; to use the electroporation as method to transform the cell; to try the aquacloning technique to assembly genic fragments with some trick: elongation and ligation; to have the Tecan machinery or a multi-plate reader, which automatizes the measurement of the fluorescence.

Interpretation of the results

From the data we obtained by the Tecan analysis we understand that a communication system based on quorum sensing molecules really works (it’s cool to study it in the books but it’s cooler to see the operative system at work!).
The fluorescence emitted by the system 1 with the GFP is more explicative of the oscillator pathway than the linear growth of the RFP’s fluorescence in system 2. According to the oscillation of the graphics we are inclined to think that the system 1 was fitting better the oscillatory system and perhaps this is confirmed by the linear increase of RFP’s emission. Indeed, the constant growth of red light could signify that system 2 experiences a basal level of induction. On the other hand looking at the comparative graphs of the ratio of the fluorescence with the two strains, it is clearly that the presence of tetracycline in the cultivation media induce a bigger production of RFP than the production of GFP. This last consideration could be in agreement with the first one because if the RFP’s system has a basal level of induction the genic oscillator with his cycle of induction and inhibition participates to increase the production of RFP.

Successful results:

Obtaining recombinant cells transformed with K876011 plasmid through electroporation;
Observation in the electrophoretic run the presence of a genic fragment assembled by acquacloning method;
Obtaining fluorescence data with Tecan analysis.
We did some attempts to do the acquacloning: finally with elongation and ligation we obtain the entire genic sequence;
We created a software for the light-to-sound conversion.

Failure results:

The thermal shock transformation didn’t work in summer because of the high temperature in the lab;
Many failed PCRs: we tried many times this reaction to check the best annealing temperature to amplificate the IDT’s fragment.;
We were not able to lengthen a fragment using the primers in the PCR. When we constructed virtually the system 3 we had to cut it in 5 fragments to have them synthesized by IDT; the third fragment had to be shorter of 40bp to be made, so we designed it shorter thinking to extend it with PCR’s special primers;
Because of the previous problem, we couldn’t construct our third system;
Gibson Assembly reaction didn’t work on fragments of system 3;
We were not able to recombine the system 1 fragment in the iGEM vector pSB1C3 both with blunt ligation and restriction enzyme ligation; to do the final experiment we used the genic insert recombined in pJET vector.

Plate disposition

Tecan analysis

GFP reads

RFP reads

Team:UNIFI/Results