Difference between revisions of "Part:BBa K415006"

Latest revision as of 01:40, 27 October 2010

pLux/cI-OR : RBS-mCherry : Term : p(tetR) : RBS-luxR : Term

This composite is composed of the parts Part:BBa_K415000 and Part:BBa_S03119. It can produce both luxR and mCherry. The production of luxR is normally constitutive, but can be repressed by TetR and derepressed by aTc. The production of mCherry is activated by c6-AHL and luxR, and repressed by cI. This is a test part that generates a reporter.

Confirmation: The part was constructed via the insertion of BBa_S03119 into BBa_K415000 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. NcoI and SphI enzymes were used for the restriction mapping, resulting in bands of 1089 base pairs and 2970 base pairs in agreement with sequencing predictions. The part was also sequenced and confirmed in that way.

Figure 1

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

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 +/- 130uM AHL and Amp antibiotic in a 37C incubator with dh5-alpha cells. 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 with LuxR. The fluorescence of mCherry was measured in arbitrary units.

Figure 3

Note that the chart is plotted against a log scale. With AHL induction, mCherry fluorescence increases nearly 433%. The following are FACs sheets quantifying the results.

Figure 4: K415006 -AHL

Figure 5: K415006 +AHL

The quadrants are scaled so that the auto-fluorescence of dead cells are confined within quadrant 3. BBa_K415006 has been co-transformed with one of the Collins' toggles, pTSMa, which has since been biobricked as Part:BBa_K415300 from [http://www.pnas.org/content/101/22/8414.full Collins/Kobayashi UV Toggle] (2004). It is a bistable toggle (on or off state) and switching to state 1 is induced by UV exposure and to state 2 is IPTG. If the toggle is set with IPTG, the cells will express cI which will inhibit Plambda. If this is exposed to certain levels of UV (see power modulations) cI is cleaved by Rec-A (a UV induced enzyme) and lacI expression begins and inhibits Ptrc. The samples of pTSMa/K415006 were grown overnight in 4ml of LB with 3 mM IPTG with +/- 130uM AHL and Amp antibiotic in a 37C incubator. Cells were then pelleted and washed twice, each time centrifuging at 13000 rpm for 10 minutes. 100ul of cells are resuspended and added on top of 4ml of M9 top agar with appropriate antibiotic in a mini petri dish. Cells were then UV exposed at 80 J/m^2. Cells were then pipetted up and regrown in 2ml of LB, appropriate antibiotics, and +/-AHL.

Figure 6

The change in fluorescence amounts to a nearly 654% increase. The decreased mCherry fluorescence in the -AHL case can be attributed to the lack of 'leaky' expression because of the inhibitory effects of cI protein synthesized by Part:BBa_K415300, resulting in a more binary circuit response to AHL induction.

The following are FACs sheets quantifying the results.

Figure 7: pTSMa/K415006 -AHL

Figure 8: pTSMA/K415006 +AHL