Team:MIT k415000



From the 2010 MIT iGEM Team.See the Parts Registry Page!! →

BBa_K415000 represents an AND-Gate promoter controlling the expression of expressing mCherry. The promoter requires two inputs: an environment free of cI and secondly, quorum sensing, in the form of LuxR-homoserine-lactone for triggering the pLux promoter. The part was constructed via the insertion of BBa_J06702 into BBa_R0065 which has the backbone pSB1A2.

The following 1% agarose gel, prepared from agarose mix and 1% TAE and stained with SYBR® Safe DNA gel stain, presents confirmation of said construction. The part was run along the standard of Hyperladder 1.

Figure 1

The following is a plasmid map of the part in the pSB1A2 backbone:

Figure 2

The fluorescence of mCherry was then quantified using FACs(Fluorescence-activated cell sorting) running two samples: -AHL and +AHL. The samples were grown overnight in 4ml of LB with 3 mM IPTG with +/- 130uM AHL and Amp antibiotic in a 37C incubator.
Plasmid pRCV3(Plux-GFP) was used as control to ensure adequate concentrations of AHL induction. The addition of AHL tests whether or not the circuit will respond without LuxR. The fluorescence of mCherry was measured in arbitrary units.

Figure 3

It is clear from the above figure that the average mCherry fluorescence and thus this circuit does not respond to the addition of AHL. The following are FACs sheets:

Figure 4: K415000 -AHL

Figure 5: K415000 +AHL

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