Difference between revisions of "Part:BBa K2558214"
 
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<partinfo>BBa_K2558214 short</partinfo>
 
<partinfo>BBa_K2558214 short</partinfo>
  
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This construct is the NEON positive feedback device, containing constantly expressed luxR, lux pR-HS (K2258001) controlled luxI, and lux pR-HS (K2258001) controlled GFP. Without AHL stimulation, the lux pR-HS promotor is repressed by the dCas9-gRNA expressed by Safety Catch system. Therefore, the luxI and GFP cannot be expressed. Upon the stimulation of AHL, the CRISPRi system in Safety Catch is inhibited, thus the lux pR-HS promotor is exposed, and then luxR-AHL complex now can bind and activate lux pR-HS promotor. Thereofre, luxI and GFP can now be expressed. Besides, this system will keep producing more luxI-AHL, which will form a positive feedback and increase the expression of GFP.  
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This construct is the NEON positive feedback device, containing constantly expressed luxR, lux pR-HS (K2258001) controlled luxI, and lux pR-HS (K2258001) controlled GFP. Without AHL stimulation, the lux pR-HS promotor is repressed by the dCas9-gRNA expressed by Safety Catch system. Therefore, the luxI and GFP cannot be expressed. Upon the stimulation of AHL, the CRISPRi system in Safety Catch is inhibited, thus the lux pR-HS promotor is exposed, and then luxR-AHL complex can now bind to and activate lux pR-HS promotor. Therefore, luxI and GFP can now be expressed. Besides, this system will keep producing more luxI-AHL, which will form a positive feedback and increase the expression of GFP.  
  
 
===Usage and Biology===
 
===Usage and Biology===
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The NEON system, including Neon the positive feedback plasmid (https://parts.igem.org/Part:BBa_K2558214), and Safety Catch the CRISPRi plasmid (https://parts.igem.org/Part:BBa_K2558215, https://parts.igem.org/Part:BBa_K2558216), is expected to establish a positive feedback system with low leakage and high sensitivity.
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Assisted with the experiences we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid (BBa_K2558214), and Safety Catch the CRISPRi plasmid (BBa_K2558215, BBa_K2558216) work together.
  
 
===Results===
 
===Results===
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We are still calibrating the NEON system. The results are preliminary, however from Figure.1 we can conclude that the system works to some extent. The positive feedback plasmid Neon-HS has the highest expression due to uncontrollable leakage. Original lux pR and the new lux pR-HS we designed had lower basal expression. The addition of Safety Catch and Safety Catch-HS can almost eliminate any leakage. It is foreseeable that with appropriate parameters NEON system can be activated to 104 fold.
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We are still calibrating the NEON system. The results are preliminary, however from Figure.1 we can conclude that the system works to some extent. The positive feedback plasmid Neon (BBa_K2558214) had the highest expression due to uncontrollable leakage. Original lux pR (BBa_R0062) and the new lux pR-HS (BBa_K2558001) we designed had lower basal expression. The addition of Safety Catch (BBa_K2558215) and Safety Catch-HS (BBa_K2558216)  almost eliminated the leakage of both positive feedback and the non-positive feedback systems. It is foreseeable that with appropriate parameters NEON system can be activated to almost 10^4 fold.
  
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<br/><li style="display: inline-block;"> [[File:T--Tsinghua-BasalExpressionOf LuxQuorumSensingSystems.png|thumb|center|600px|'''Figure.1. T--Tsinghua-BasalExpressionOf LuxQuorumSensingSystems.''' The fluorescence intensity is measured by flow cytometer at 488 nm. Neon-HS, Safety Catch, Safety Catch-HS and lux pR, lux pR-HS test plasmids are transferred into E.coli DH5α. The higher the fluorescence is, the more basal expression the system has.]]  
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<br/><li style="display: inline-block;"> [[File:T--Tsinghua-BasalExpressionOf LuxQuorumSensingSystems.png|thumb|center|600px|'''Figure.1. Basal expression of lux quorum sensing systems.''' The fluorescence intensity was measured by flow cytometer at 488 nm. Neon, Safety Catch, Safety Catch-HS and lux pR, lux pR-HS test plasmids were transferred into ''E.coli'' DH5α. High fluorescence intensity suggests high level of basal expression in the system. ]]  
 
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===Protocol===
 
===Protocol===
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# Transform the plasmids into E. coli DH5α according to the design in Table 2.
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# Transform the plasmids into ''E. coli'' DH5α.
 
# Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.  
 
# Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.  
 
# Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
 
# Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
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# Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Use flow cytometry to measure fluorescent intensity in 1.5 mg/ml Kanamycin treated medium at one hour intervals.
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# Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
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# Each group is repeated for at least 3 times.
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# Each group should be repeated for at least 3 times.
  
  
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<br/>[2]Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., & Arkin, A., et al. (2013). Repurposing crispr as an rna-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173.
 
<br/>[2]Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., & Arkin, A., et al. (2013). Repurposing crispr as an rna-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173.
  
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====Sequence and Features====
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===Sequence and Features===
 
<partinfo>BBa_K2558214 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2558214 SequenceAndFeatures</partinfo>
  

Latest revision as of 16:42, 17 October 2018


Neon lux positive feedback device with luxpR-HS promotor

This construct is the NEON positive feedback device, containing constantly expressed luxR, lux pR-HS (K2258001) controlled luxI, and lux pR-HS (K2258001) controlled GFP. Without AHL stimulation, the lux pR-HS promotor is repressed by the dCas9-gRNA expressed by Safety Catch system. Therefore, the luxI and GFP cannot be expressed. Upon the stimulation of AHL, the CRISPRi system in Safety Catch is inhibited, thus the lux pR-HS promotor is exposed, and then luxR-AHL complex can now bind to and activate lux pR-HS promotor. Therefore, luxI and GFP can now be expressed. Besides, this system will keep producing more luxI-AHL, which will form a positive feedback and increase the expression of GFP.

Usage and Biology

Assisted with the experiences we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid (BBa_K2558214), and Safety Catch the CRISPRi plasmid (BBa_K2558215, BBa_K2558216) work together.

Results

We are still calibrating the NEON system. The results are preliminary, however from Figure.1 we can conclude that the system works to some extent. The positive feedback plasmid Neon (BBa_K2558214) had the highest expression due to uncontrollable leakage. Original lux pR (BBa_R0062) and the new lux pR-HS (BBa_K2558001) we designed had lower basal expression. The addition of Safety Catch (BBa_K2558215) and Safety Catch-HS (BBa_K2558216) almost eliminated the leakage of both positive feedback and the non-positive feedback systems. It is foreseeable that with appropriate parameters NEON system can be activated to almost 10^4 fold.


  • Figure.1. Basal expression of lux quorum sensing systems. The fluorescence intensity was measured by flow cytometer at 488 nm. Neon, Safety Catch, Safety Catch-HS and lux pR, lux pR-HS test plasmids were transferred into E.coli DH5α. High fluorescence intensity suggests high level of basal expression in the system.

    Protocol

    1. Transform the plasmids into E. coli DH5α.
    2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
    3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
    4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
    5. Each group should be repeated for at least 3 times.


    Reference

    [1]Afroz, T., & Beisel, C. L. (2013). Understanding and exploiting feedback in synthetic biology. Chemical Engineering Science, 103(11), 79-90.
    [2]Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., & Arkin, A., et al. (2013). Repurposing crispr as an rna-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173.


    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal NheI site found at 2026
      Illegal NheI site found at 2049
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 801
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 950
      Illegal SapI.rc site found at 1129