Difference between revisions of "Part:BBa K415069"

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<partinfo>BBa_K415069 short</partinfo>
 
<partinfo>BBa_K415069 short</partinfo>
  
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This is a LuxR/LacO-regulated GFP, LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification, TetR-regulated LuxR production.
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This is a LuxR/LacO-regulated GFP, LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification, TetR-reguan improvement on [[Part:BBa_K415023]]. The promoter controlling LuxI expression is [[Part:BBa_K415031]], a less leaky version of [[Part:BBa_R0065]]. This modification significantly reduces the noise caused by the original promoter driving luxI production, and thus auto-induction.
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This is nearly the same part as [[Part:BBa_K415023]] except that the promoter controlling LuxI expression is [[Part:BBa_K415031]], a less leaky version of [[Part:BBa_R0065]].
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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_K415032]]. 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) or along with an improved low-power toggle switch Part:BBa_K415301, which requires significantly less(10 times less) UV exposure for activation.
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The co-transformation of Part:BBa_K415301 with BBa_K415069 form a system for UV inducible precision pattern formation with exposure times under a second. Part:BBa_K415301 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.
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K415069 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K415069 SequenceAndFeatures</partinfo>
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The following is the plasmid map of BBa_K415069
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Figure 1:
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[[Image:069plasmid.png|center|800px]]
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== FACs Data ==
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The fluorescence of mCherry and GFP was then quantified using FACs(Fluorescence-activated cell sorting).
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The samples of pTSMa/K415069 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.
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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. This was done instead of simply plotting the mean fluorescence because of the bimodal fluorescence distributions in many of the samples, likely a result of cell death from UV exposure.
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Note that TSM stands for pTSMa ([[Part: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.
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[[Image:Sdm.png|left|frame|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.]][[Image:Deathcurve.png|thumb|right|E.Coli population density as a function of UV exposure. Measured by drip assay.]]
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Figure 6: % Cells satisfying threshold set for mCherry fluorescence.
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[[Image:mCherry.png‎|center|600px]]
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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. From Figure 6, we observe that that replacing R0065 with [[Part:BBa_K415032]] results in a reduction of 'leaky' mCherry fluorescence by up to 60%. This means better control over cell scale fluorescence for tighter, more precise patterns.
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Figure 7: % Cells satisfying threshold for GFP fluorescence. Note that 069 represents [[Part:BBa_K415069]], an improvement on BBa_K415023 by reducing the 'leakiness' of the R0065 promoter in front of the luxI.
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[[Image:K415023 GFP.png‎|center|600px]]
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The Figure 7 presents 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]]).
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Moreover, from the GFP curve, we can immediately see the improvements garnered by the new promoter driving luxI. Comparing between parts with the same toggle switch, there is a nearly 60% decrease in auto-induction GFP background fluorescence. This significant reduction of noise opens the possibility of precision pattern and potentially, biomaterial 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.
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Figure 8: pTSMa ([[Part:BBa_K415300]])/K415069
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[[Image:p069 0.png|left|thumb|0 UV]] [[Image:p069 3.png|left|thumb|3 UV]] [[Image:p069 5.png|left|thumb|5 UV]] [[Image:p069 10.png|left|thumb|10 UV]]
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[[Image:p069 20.png|left|thumb|20 UV]][[Image:p069 30.png|left|thumb|30 UV]]    [[Image:p069 40.png|left|thumb|40 UV]]
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Figure 9: pLPTa ([[Part:BBa_K415301]])/K415069
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[[Image:l069 0.png|left|thumb|0 UV]]
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[[Image:l069 3.png|left|thumb|3 UV]]
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[[Image:l069 5.png|left|thumb|5 UV]]
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[[Image:l069 10.png|left|thumb|10 UV]]
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[[Image:l069 20.png|left|thumb|20 UV]][[Image:l069 30.png|left|thumb|30 UV]]  [[Image:l069 40.png|left|thumb|40 UV]]
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Latest revision as of 17:46, 27 October 2010

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

This is a LuxR/LacO-regulated GFP, LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification, TetR-reguan improvement on Part:BBa_K415023. The promoter controlling LuxI expression is Part:BBa_K415031, a less leaky version of Part:BBa_R0065. This modification significantly reduces the noise caused by the original promoter driving luxI production, and thus auto-induction. 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_K415032. 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) or along with an improved low-power toggle switch Part:BBa_K415301, which requires significantly less(10 times less) UV exposure for activation. The co-transformation of Part:BBa_K415301 with BBa_K415069 form a system for UV inducible precision pattern formation with exposure times under a second. Part:BBa_K415301 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 3687
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 747
    Illegal BsaI.rc site found at 2954


The following is the plasmid map of BBa_K415069

Figure 1:

069plasmid.png


FACs Data

The fluorescence of mCherry and GFP was then quantified using FACs(Fluorescence-activated cell sorting). The samples of pTSMa/K415069 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. This was done instead of simply plotting the mean fluorescence because of the bimodal fluorescence distributions in many of the samples, likely a result of cell death from UV exposure. Note that TSM stands for pTSMa (Part: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.

MCherry.png


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. From Figure 6, we observe that that replacing R0065 with Part:BBa_K415032 results in a reduction of 'leaky' mCherry fluorescence by up to 60%. This means better control over cell scale fluorescence for tighter, more precise patterns.


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

K415023 GFP.png


The Figure 7 presents 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). Moreover, from the GFP curve, we can immediately see the improvements garnered by the new promoter driving luxI. Comparing between parts with the same toggle switch, there is a nearly 60% decrease in auto-induction GFP background fluorescence. This significant reduction of noise opens the possibility of precision pattern and potentially, biomaterial 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.



Figure 8: pTSMa (Part:BBa_K415300)/K415069

0 UV
3 UV
5 UV
10 UV













20 UV
30 UV
40 UV















Figure 9: pLPTa (Part:BBa_K415301)/K415069

0 UV
3 UV
5 UV
10 UV













20 UV
30 UV
40 UV