Part:BBa_K415023

pLux-Lac:GFP:Term:Term:PluxR/cI-OR :mCherry:Term:PtetR:LuxR:Term:Plux/cI-OR:LuxI

This is a LuxR/LacO-regulated GFP, LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification, TetR-regulated LuxR production. This part displays red and green fluorescence under specific conditions. Fluorescence is activated by presence of both LuxR and 3OC6HSL. The LuxR is normally constitutively expressed, but can be repressed by TetR and derepressed by aTc. Green fluorescence is repressed by LacI and red fluorescence and auto-induction is repressed by CI.This composite is composed of the parts Part:BBa_K415021 and Part:BBa_K415019. It can produce GFP, luxR, mCherry, and luxI and is thus auto-inducing. The production of luxR is normally constitutive, but can be repressed by TetR and derepressed by aTc. The production of mCherry and luxI are activated by c6-AHL and luxR, and repressed by cI. This part is intended to be used along with one of the Collins' toggles, pTSMa, which has since been biobricked as Part:BBa_K415300 from 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3716
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 747

The following is the plasmid map of the part:

Figure 1

Images

The following are confocal microscope images of pTSMa/K415023 after varying UV exposure levels.

The samples of pTSMa/K415023 co-transformed in JM2.300 cells were grown overnight in 4ml of LB with 3 mM IPTG with Amp and Kan antibiotic in a 37C incubator. Cells were then pelleted and washed twice, each time centrifuging at 13000 rpm for 10 minutes. 400ul of cells are resuspended and embedded in of 4ml of M9 top agar with appropriate antibiotic in a mini petri dish. Cells, once in the plates, were then incubated for an additional 3 hours at 30C. Cells were then UV exposed at varying intensities and incubated for another 3 hours at 30C.

Experimental setup for UV exposure. Exposed pattern of star approximately 1 inch from opposite tips.

Apparatus for UV exposure.

Figure 2: pTSMa/K415023. Varying UV Exposure Levels. Increases from bottom right to top left, 0 to 80 J/m^2

Figure 3: Comparison of Part:BBa_K415300 and Part:BBa_K415301 toggles with BBa_K415023 reporter

Figure 4: pTSMa/K415023. Settings set for mCherry detection.

Figure 5: pTSMa/K415023. mCherry expressed in UV exposed regions. GFP background.

The fluorescence of mCherry and GFP was then quantified using FACs(Fluorescence-activated cell sorting). The samples of pTSMa/K415023 co-transformed in JM2.300 cells were grown overnight in 4ml of LB with 3 mM IPTG with Amp and Kan 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 varying intensities. Afterwards, cells were pipetted up and regrown in 2ml of LB, appropriate antibiotics. Plasmid pRCV3(Plux-GFP) and pINV5(pLacIQ->lacI; pLac->GFP) were used as control to ensure adequate concentrations of AHL and IPTG induction. The following graphs plot the percentage of cells that satisfy a threshold of fluorescence of mCherry and GFP versus UV exposure. The threshold was set just above the auto-fluorescence of dead cells. Note that TSM stands for pTSMaPart:BBa_K415300 and that LPT represents an improved toggle in comparison to its predecessor Part:BBa_K415300 in that it requires less UV power to switch states, thus killing fewer cells (see E.Coli death curve). This plasmid differs genetically from Part:BBa_K415300 in that its inhibitory lambda cI protein is more sensitive to Rec-A cleavage. We got the idea for hypersensitive cI from [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC218967/pdf/jbacter00221-0157.pdf this paper] which calls for a single point mutation in the protein. By changing the Glu233->Lys, we were able to create a toggle that is more sensitive to UV induction. We accomplished this mutation through site directed mutagenesis.

The site directed mutagenesis that the 2010 MIT iGEM team performed on the Collins toggle pTSMa in order to change it into a Low Power Toggle.

E.Coli population density as a function of UV exposure. Measured by drip assay.

Figure 6: % Cells satisfying threshold set for mCherry fluorescence.

Figure 7: % Cells satisfying threshold for GFP fluorescence. Note that 069 represents Part:BBa_K415069, an improvement on Part:BBa_K415023 by reducing the 'leakiness' of the R0065 promoter before the luxI.

The curves demonstrate a high GFP background expression during the inoculation stage with IPTG in culture, independent of UV radiation because of the slow degradation period of the GFP(Part:BBa_E0040). However, mCherry expression is directly correlated with UV exposure levels. UV exposure triggers the E. coli SOS response, cleaving the cI proteins and resulting in the anti-inhibition of auto-induction from luxI and mCherry, both driven by the R0065 luxR-cI hybrid promoter.