Even though the assembling of the parts gave us an big headache and end up not working, the PCR products worked fine. We managed to amplify the genes we intended to use without any problems.
Electrophoresis of PCR products.Source: Personal archive.
The assemblies of the biobricks worked out all right. It seems to us that something went wrong specifically with the Gibson Assembly, because all the iGEM protocols seemed to work.
Another sign of positive results is the assembly of the operon, which we already stated didn't work, when we used 3A assembly to join the Lux parts, the product came out just fine, even tough it worked too close to the due date, so we did not have time to experiment or even caracterize the new part.
Even tough we managed to assemble the Lux Operon, we had not enough time to express it and much less try to biosynthesize our beloved alkanes. So we ended up expressing only the LuxD gene.
We assembled a plasmid with the LuxD gene and a RFP gene to ensure a visual representation of our expression. And the grown bacteria expressed quite a lot of the gene.
Pelletized cells.Source: Personal archive.
So with our visual expression confirmation, we went for the actual confirmation. We then digested our cells and separated our gene from it's plasmid. With our product had both the RFP and the LuxD gene in it's cut product, the relation between the RFP protein and the LuxD protein expressions are then proved.
And our gel product proved to be also really intense, with well defined and strong bands. Analyzing the band size, it is also pretty clear the gene composition of it, with around 1740bp for the LuxD gene and 905bp for the RFP, our expression should turn out somewhere between the 2000bp ant 3000bp band, with 2645bp.
Electrophoresis from expressed LuxD+RFP, with Quick-Load Purple 2-log DNA ladder .Source: Personal archive.