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===
 
===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.
 
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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<center>Figure7</center>
 
<center>Figure7</center>
  
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GFP gene is exchanged for dCas9 with companion of constantly-expressed single guide RNA to establish the qs-CRISPRri system. Through the donor-receiver system, we realized spatial-level control of cell growth (Figure 8). Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.
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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.
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<center>https://2019.igem.org/wiki/images/e/e0/T--Peking--8.jpg</center>
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<center>Figure8</center>
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Furthermore, a complete quorum sensing system-implanted strain is tested with microfluidic system. Distinct from arabinose-induced CRISPRri system, decrease in cell division rate shows an ultrasensitive behavior. The cells were found to almost stop division and only lengthen its own shape all of a sudden, which we believe to be attributed to accumulation of AHL. Since cell growth is exponential at early stage, AHL concentration would increase rapidly. As AHL concentration reaches a certain threshold, cell genome replication is interrupted by binding of dCas9 and cell cycle extends largely. This enables E. coli to rapidly autoregulates its own growth state, and provides a potential concept of self-tuning medical-used bacteria without external interference.
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We hope to construct a self-regulating system, but in our experiment, the expression of LuxI is still artificially induced by IPTG. The only reason why we do not use a constant promoter is that we try to avoid over-inhibition of cell growth during molecular cloning. In fact, it can be replaced by any constant promoters with proper strength to realize full-automatic regulation of growth in real application.
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Revision as of 15:56, 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
Figure7

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.