Team:TUDelft/Results

Results

The first results obtained were the successfully assemblies of the plasmids 1 (BBa_K175046) and 2 (BBa_K175047) (see the plan). [As showed in the gel image, the bands are present in the proper lengths]. After corroborate the length of the new biobricks, we preceded to their electro transformation in Escherichia coli TOP10 cells (see protocols). Besides, we send samples of miniprep DNA to sequence; we were able to confirm the presence of the last part of the biobrick (RBS + GFP + Terminator). Further investigation on the sequence should be done.

250 µL of the E. coli TOP10 cells carrying either the plasmid 1 or 2 were plated in an LB agar Petri dish adding the proper selection antibiotic and incubated at 37ºC overnight. The colonies in the plate for plasmid 1 showed red fluorescence and the colonies in the plate for plasmid 2 showed green fluorescence (data not shown). These results were in agreement with the expected fluorescence, Ptet (R0040) act as a constitutive promoter in the absence of its repressor TetR (C0040), therefore, the production of cI (C0051) and RFP (E1010) were present in the cells. Similar explanation could be used for the interpretation of the green glow in plasmid 2.

The delay-device is formed by the two plasmids. Therefore we introduce both plasmids into the TOP10 cells. We plated and cultured them using the same conditions mentioned above. In this case the plates and culture tubes were glowing red, which is in agreement with our expectations (see figure XXX). However in preliminary tests (data not shown), the addition of IPTG to the medium did not lead to the change of fluorescence from red to green as expected. These preliminary results indicate the lack of function of the plasmid 2 when the both plasmids are present. This conclusion is only accepted if the presence of the two plasmids in one cell is confirmed, however we could not confirm it; thus it will remain as another potential explanation.

Derived from the results in other sections of this subproject (see XXXX), the plasmids were also transformed in (K12) electro-competent cells. 250 µL of the E. coli (K12) cells carrying either the plasmid 1, plasmid 2 or both were plated in an LB agar Petri dish adding the proper selection antibiotic and incubated at 37ºC overnight. As the previous results, the plates with cells containing the plasmid 1 and the colonies carrying both plasmids glowed red and the cells with the plasmid 2 glowed green. One colony from each plate was inoculated in a 4 mL culture tube with minimal medium and the appropriate antibiotic. The tubes were incubated overnight at 37°C and 175 rpm. Once again, the expected results were confirmed (se figure XXX).

The tube containing cells with both plasmids were used to perform a simple kinetic in order to have preliminary results for the delay time. 1mL was transfer to a tube with 4mL fresh minimal medium and incubated for 1 h at 37°C and 175 rpm. This procedure was done twice in order to have a control. After 1 h one tube was induced with 0.2 mM IPTG and the glowing was followed for 10 h (see figure XXX).

No change of color was noticed and the red glow was increasing over time indicating the growth of the culture. Unfortunately, these results proved once again the non functionality of the device under the circumstances tested. Further experiments, see below, were performed with some promising results.

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