Difference between revisions of "Part:BBa K415022"
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<partinfo>BBa_K415022 short</partinfo>
 
<partinfo>BBa_K415022 short</partinfo>
  
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This is a LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification and TetR-regulated LuxR production. This composite is composed of the parts BBa_K415012([[Part:BBa_K415006]] on low copy number backbone) and [[Part:BBa_K415019]]. It can produce 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 is a test part that generates a reporter.
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This is a LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification and TetR-regulated LuxR production. This composite is composed of the parts BBa_K415012 ([[Part:BBa_K415006]] in the low copy number backbone [[Part:pSB3K3]]) and [[Part:BBa_K415019]]. It can produce 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 is a test part that generates a reporter.
  
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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_K415022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K415022 SequenceAndFeatures</partinfo>
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Confirmation: The part was constructed via the insertion of [[Part:BBa_K415019]] into BBa_K415013([[Part:BBa_K415006 in a low copy backbone) which has the backbone pSB3K3. 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. BglII, PvuII enzymes were used for the restriction mapping, resulting in bands of 2000 base pairs and 3500 base pairs in agreement with sequencing predictions. The band at 5500 base pairs represents undigested DNA. The part was also sequenced and confirmed in that way.
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Figure 1
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[[Image:K415022 digest.jpg|center|250px]]
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The following is the plasmid map of BBa_K415022 in the [[Part:pSB3K3] backbone.
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Figure 2
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[[Image:K415022 plasmid.png|center|800px]]
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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 JM2.300 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. Induction of AHL to BBa_K415022 results in a 138% increase in mCherry fluorescence. Note that this increase is substantially less than that of [[Part:BBa_K415006]] which was an intermediate construct to BBa_K415022 that lacked the  appended Plux/cI-OR : RBS : LuxI. However, comparing the -AHL samples of BBa_k415022 and BBa_K415013 which is [Part:BBa_K415006]] on the same low copy backbone as BBa_k415022, the auto-induction by luxI in BBa_K415022 results in a 358% increase in mCherry fluorescence.
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Figure 3
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[[Image:K415022.png|center|500px]]
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BBa_K415022 has also been co-transformed 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. The samples of pTSMa/K415022 were grown overnight in 4ml of LB with 3 mM IPTG with +/- 130uM AHL and 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 60 J/m^2. Cells were then pipetted up and regrown in 2ml of LB, appropriate antibiotics, and +/-AHL. AHL induction results in a 134% increase in mCherry fluorescence for the pTSMa/K415022 co-transform. The overall shift downwards in fluorescence in comparison to BBa_K415022 alone may be attributed to the inhibitory effects of the cI proteins synthesized by the toggle during cell inoculation.
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The quadrants on the following FACs data sheets are scaled so that the auto-fluorescence of dead cells are confined within quadrant 3:
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Figure 4: K415022 -AHL
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[[Image:K415022+AHL.png|center|500px]]
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Figure 5: K415022 +AHL
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[[Image:K415022+AHL.png|center|500px]]
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FACs Sheets for pTSMa/K415022 after exposure to 60 J/m^2 UV radiation:
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Figure 6: pTSMa/K415022 -AHL
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[[Image:PTSMa.K415022-AHL.png‎|center|500px]]
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Figure 7: pTSMa/K415022 +AHL
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[[Image:PTSMa.K415022+AHL.png‎|center|500px]]
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Revision as of 03:02, 27 October 2010

PluxR/cI-OR : RBS : mCherry : Term : PtetR : RBS : LuxR : Term : Plux/cI-OR : RBS : LuxI

This is a LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification and TetR-regulated LuxR production. This composite is composed of the parts BBa_K415012 (Part:BBa_K415006 in the low copy number backbone Part:pSB3K3) and Part:BBa_K415019. It can produce 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 is a test part that generates a reporter.


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 2748
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Confirmation: The part was constructed via the insertion of Part:BBa_K415019 into BBa_K415013([[Part:BBa_K415006 in a low copy backbone) which has the backbone pSB3K3. 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. BglII, PvuII enzymes were used for the restriction mapping, resulting in bands of 2000 base pairs and 3500 base pairs in agreement with sequencing predictions. The band at 5500 base pairs represents undigested DNA. The part was also sequenced and confirmed in that way.

Figure 1

K415022 digest.jpg


The following is the plasmid map of BBa_K415022 in the [[Part:pSB3K3] backbone.

Figure 2

K415022 plasmid.png


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 JM2.300 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. Induction of AHL to BBa_K415022 results in a 138% increase in mCherry fluorescence. Note that this increase is substantially less than that of Part:BBa_K415006 which was an intermediate construct to BBa_K415022 that lacked the appended Plux/cI-OR : RBS : LuxI. However, comparing the -AHL samples of BBa_k415022 and BBa_K415013 which is [Part:BBa_K415006]] on the same low copy backbone as BBa_k415022, the auto-induction by luxI in BBa_K415022 results in a 358% increase in mCherry fluorescence.

Figure 3

K415022.png

BBa_K415022 has also been co-transformed 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. The samples of pTSMa/K415022 were grown overnight in 4ml of LB with 3 mM IPTG with +/- 130uM AHL and 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 60 J/m^2. Cells were then pipetted up and regrown in 2ml of LB, appropriate antibiotics, and +/-AHL. AHL induction results in a 134% increase in mCherry fluorescence for the pTSMa/K415022 co-transform. The overall shift downwards in fluorescence in comparison to BBa_K415022 alone may be attributed to the inhibitory effects of the cI proteins synthesized by the toggle during cell inoculation.

The quadrants on the following FACs data sheets are scaled so that the auto-fluorescence of dead cells are confined within quadrant 3:

Figure 4: K415022 -AHL

K415022+AHL.png


Figure 5: K415022 +AHL

K415022+AHL.png


FACs Sheets for pTSMa/K415022 after exposure to 60 J/m^2 UV radiation:

Figure 6: pTSMa/K415022 -AHL

PTSMa.K415022-AHL.png


Figure 7: pTSMa/K415022 +AHL

PTSMa.K415022+AHL.png