Difference between revisions of "Part:BBa K2033001:Design"
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===Stage 1-Insert Regulator ORFs===
 
===Stage 1-Insert Regulator ORFs===
 
====Phusion PCR====  
 
====Phusion PCR====  
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The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found [https://benchling.com/hayneslab/f/rvhDwTUE-older/seq-nAjbajRk-modular_receiver_annotated/edit here]. The following reaction was conducted to amplify and fuse AubR with DNA plasmid.
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The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found [https://benchling.com/hayneslab/f/rvhDwTUE-older/seq-nAjbajRk-modular_receiver_annotated/edit here.] The following reaction was conducted to amplify and fuse AubR with DNA plasmid.
  
 
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Revision as of 19:49, 13 September 2016


C(12)-HSL Receiver Device - AubR


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 395
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 395
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 395
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 395
    Illegal AgeI site found at 205
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 184


Design Notes

Here is the inducible promoter sequence:

Stage 1-Insert Regulator ORFs

Phusion PCR

The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found here. The following reaction was conducted to amplify and fuse AubR with DNA plasmid.

Phusion PCR Reaction
Reagent Volume(uL) Mix(x5) Thermal Cycler Step Step Temperature (°C) Step Length (sec) Step Repeats
DNA Plasmid 1.0 5.0 Initial Denaturation 98 180 1x
10uM F Primer 1.0 5.0 Denaturation 98 10 35x
10uM R Primer 1.0 5.0 Annealing 66 30 35x
10mM dNTPs 1.0 5.0 Elongation 72 60 35x
Phusion Pol. 0.5 2.5 Elongation 72 600 1x
5x HF buffer 10 50 Cold Storage 4 ∞ 1x
dH2O 36 180
Total 50.5

Stage 2-Insert Promoters

Source

This HSL receiver was recovered from a metagenomic project on soil. The specific source is unknown.

References

(1) Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. "Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach." Applied and Environmental Microbiology 78.22 (2012): 8067-074. Web