Demonstrate

Step 1

The goal of our science-art project is to obtain music from Escherichia coli and in order to achieve that we decided to engineer our E.coli strains with an oscillating circuit dependant on quorum sensing molecules and able to produce two fluorescent proteins (GFP and RFP) to form patterns of oscillating fluorescence. As oscillating circuit we used the population toggle formed by the two parts BBa_K876008 and BBa_K876011 made by iGEM12 UT Dallas Team and we engineered E.coli with those plasmids in order to have two populations with one plasmid each and able to “talk” each other thanks to the feedbacks provided by quorum sensing molecules (AI-1 and AHL).
Depending on the concentrations of those molecules, the pathways leading to fluorescent proteins expression are alternately switched on, and this leads to rapid changes in the main colour of fluorescence and also grants a constant variation of the intensity of each fluorescence.

Step 2

Fluorescence colour and intensity can easily be read with the automated reader Tecan InfinitePro200, a machinery well-tested with references in over 1800 peer-reviewed publications. The reader uses 96-well plates to measure the fluorescence of each well in which we put our LB cultures with the two populations together; moreover, to attain different reads we used a variety of ratio between the two populations and different concentrations from well to well of the two inducers tetracycline and IPTG.

Step 3

The results obtained with Tecan reader are excel files reporting excitation and emission wavelength, bandwidth of emission and values of fluorescence intensity over time (i.e. each box of the matrix represents the value of measured fluorescence at a different points of bacteria’s growth). It is clear that the most intense fluorescence is the one given by GFP, since it is the most stable molecule, and this is represented by the base gap of 30.000 units of fluorescence between the two. Nonetheless we normalized the results so that the software could read every value with the same starting point.

Step 4

The XLS files collected can then be converted into a musical melody thanks to our software Bachteria; the software is based on a simple sequence of processes, from reception to rendering, and in each step many pieces of code make their own part in the translational system. The ratio based on which the software works has been elaborated by integrating in our project the aid of a musician and thanks to him the software ensemble can now produce complex melodies.

Software compatibility:

The software is open to the public and part of the codes are under free license; nonetheless anyone willing to use it should take note that the XLS file to be uploaded in the software has to be properly fitted to be readable. The said file must have no blank boxes, since a blank box would result in the end of the translation; moreover there must be a maximum of ten column filled with values, but there are no limitations on the number of lines; also, if using different colours of fluorescence it is always best to put first the ones with the higher average intensity (e.g. in our case we put first GFP, then RFP). Excel readable example

Audio Example:

The Sound of coli

Team:UNIFI/Demonstrate