Team:UNIFI/Contribution
Contribution
In June 2017 we worked on the BBa_K876011, which we named System 2 in agree with our project.{Image 1}
We have produced a cytoplasmatic oscillator based on two different system, included in two different plasmids: the system 1 expressed GFP and it’s induced by IPTG, as first molecules; the system 2 expressed RFP and it’s induced by tetracycline. While system 1 was not present in the iGEM plates and we had to order the sequence at the IDT, the genic sequence of system 2 was in the second plate in the 19H well.
We had resuspended the dry DNA with 10µl of dH2O and used 1µl of the plasmid to transform with electroporation DH5α cells. The efficiency of the experiment was not so good (it was about 10^5 cfu/µg) but the grown colonies were pink so there were a good probability of a successful experiment! We proceeded with the isolation of the colonies in 10 plates; after the overnight growth we saw again pink colonies.{Images 2-3}
Then we saved the strain preparing a stock of glycerol. We also made a plasmid extraction to check our DNA was effectively in the cells (we controlled two different strains of the 10 isolation) and the electrophoresis run showed them.{Image 4}
In September, when we obtained the system 1 too, we have checked if the oscillator really works analysing the fluorescence in the Tecan machine.{Image 5}
The graphic shows how the red emission changes during the cycles (a cycle is a measurement, and each measure is made after a gap of 1h) in different conditions: the strain with K876011 plasmid in the LB media and IPTG produced the smallest fluorescence emission, which grows when in the media are added the IPTG+Tetracycle (the second one is the primary inductor of the single system 2) and it has bigger values in the strain in only LB media (indeed the colonies were pink), until the biggest emission we got from the strain cultivated in the LB+Tetracycline media in which there is the inductor of the system. The fluorescence values have a spike in the middle of the measurements when probably the growth achieved the plateau; infact after that there’s a reduction and a stabilization of the emission because the cell were not able any more to growth in a small volume such as the one of the well in a 96-well plates and in the case of the system induced by tetracycline we assist to a reduction also of the fluorescence, maybe caused by the negative influence the antibiotics could have on the strain.
When there is only one system in a well we do not see the very effect of the oscillator, which promptly appears in the wells with various diluition of the two strain with the two system. The oscillation of the fluorescence you can see in the result part -> link.
Team Unifi
unifi.igem@gmail.com
