Difference between revisions of "Part:BBa K3081053"
 
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===Quorum Sensing CRISPRri System Enables Spatial-level Growth Control and Ultrasensitive Autoregulation of Growth===
  
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Hence, based on the well-characterized growth control system, we constructed a quorum sensing CRISPRri system (qs-CRISPRri) to realize smart regulation of bacterial overall states.
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<partinfo>BBa_K3081053 short</partinfo>
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Quorum sensing system is widely used in gene circuit design as a sensor of cell density. As a transcription factor, LuxR senses AHL and then activate the transcript of plux. In our project, we want dCas9 to express when bacteria density is high. So we put the dCas9 gene under the control of plux, and get this composite part. And we can use golden-gate assembly methods to change the sequence of sgRNA.  
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A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system (Figure 7). The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor.
  
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https://2019.igem.org/wiki/images/b/b5/T--Peking--QS3-.png
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<center>https://2019.igem.org/wiki/images/0/0c/T--Peking--7.jpg</center>
  
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Figure1 We transfer this part in <i>E. coli</i> ,BL21(DE3), and culture them in M9 medium containing a series of AHL concentration for 6 hours. Receiver cells show remarkable dose-dependent change in length with addition of AHL.  
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Figure 1.The donor-receiver split quorum sensing system of CRISPRri system. Upper figure is the gene circuit of donor and receiver cells of qs-CRISPRri system. For the figure below, a donor-receiver split quorum sensing GFP system enables fluorescent intensity control on spatial scale. Left figure is the sketch map of how donor and receiver bacteria is dropped onto the agar plate. Right figure is the real picture of agar plate under illumination of blue light. The location of the white arrow is the donor cells. Receiver cells are marked by number one to six.
  
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https://2019.igem.org/wiki/images/7/75/T--Peking--QS6-.png
 
  
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Figure2 The Pattern diagram of Quorum sensing system.
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In this part, we can use golden-gate assembly to change the sgRNA sequence, in order to target different sites on bacterial genome. We mainly used M+ (BBa_K3081054) for the demonstration of qs-CRISPRri system. For more detailed information, see <partinfo>BBa_K3081054</partinfo>.
  
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https://2019.igem.org/wiki/images/0/01/T--Peking--QS7.png
 
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https://2019.igem.org/wiki/images/d/de/T--Peking--QS88-.png
 
  
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Figure3 We transferred this part into <i>E. coli</i>, BL21(DE3) as receptor cells, and drop or coat them on the solid medium like A. Then drop 5 μl M9 containing 0.5 Mm IPTG. Solid medium were incubated about 16h. The bacteria density in one drop showed great difference with different distances from IPTG. 
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Reference:
  
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[1]Weiqian Zeng, et al. Rational Design of an Ultrasensitive Quorum-Sensing Switch. ACS Synthetic Biology 2017 6 (8), 1445-1452.
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[2]Y. Soma, T. Hanai. Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production. Metabolic Engineering 30 (2015) 7–158.
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3081053 SequenceAndFeatures</partinfo>
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<partinfo>BBa_T9002 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3081053 parameters</partinfo>
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<partinfo>BBa_T9002 parameters</partinfo>
 
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Latest revision as of 20:15, 21 October 2019

Quorum Sensing CRISPRri System Enables Spatial-level Growth Control and Ultrasensitive Autoregulation of Growth

Hence, based on the well-characterized growth control system, we constructed a quorum sensing CRISPRri system (qs-CRISPRri) to realize smart regulation of bacterial overall states.

A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system (Figure 7). The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor.

T--Peking--7.jpg

Figure 1.The donor-receiver split quorum sensing system of CRISPRri system. Upper figure is the gene circuit of donor and receiver cells of qs-CRISPRri system. For the figure below, a donor-receiver split quorum sensing GFP system enables fluorescent intensity control on spatial scale. Left figure is the sketch map of how donor and receiver bacteria is dropped onto the agar plate. Right figure is the real picture of agar plate under illumination of blue light. The location of the white arrow is the donor cells. Receiver cells are marked by number one to six.


In this part, we can use golden-gate assembly to change the sgRNA sequence, in order to target different sites on bacterial genome. We mainly used M+ (BBa_K3081054) for the demonstration of qs-CRISPRri system. For more detailed information, see BBa_K3081054.


Reference:

[1]Weiqian Zeng, et al. Rational Design of an Ultrasensitive Quorum-Sensing Switch. ACS Synthetic Biology 2017 6 (8), 1445-1452.

[2]Y. Soma, T. Hanai. Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production. Metabolic Engineering 30 (2015) 7–158.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1004
    Illegal BsaI.rc site found at 1732