Difference between revisions of "Part:BBa K2033001:Design"
Line 11: Line 11:
 
===Stage 1-Insert Regulator ORFs===
 
===Stage 1-Insert Regulator ORFs===
 
====Phusion PCR====  
 
====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 [https://benchling.com/hayneslab/f/rvhDwTUE-older/seq-nAjbajRk-modular_receiver_annotated/edit here].  
+
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.
  
 
{| border=1
 
{| border=1
−
   |+ Caption
+
   |+ Phusion PCR Reaction
 
|-
 
|-
 
   ! Reagent
 
   ! Reagent
 
   ! Volume(uL)
 
   ! Volume(uL)
 
   ! Mix(x5)
 
   ! Mix(x5)
 +
  ! Thermal Cycler Step
 +
  ! Step Temperature (°C)
 +
  ! Step Length (sec)
 +
  ! Step Repeats
 
|-
 
|-
 
   | DNA Plasmid
 
   | DNA Plasmid
 
   | 1.0
 
   | 1.0
 
   | 5.0
 
   | 5.0
 +
  | Initial Denaturation
 +
  | 98
 +
  | 180
 +
  | 1x
 
|-
 
|-
 
   | 10uM F Primer
 
   | 10uM F Primer
 
   | 1.0
 
   | 1.0
 
   | 5.0
 
   | 5.0
 +
  | Denaturation
 +
  | 98
 +
  | 10
 +
  | 35x
 
|-
 
|-
 
   | 10uM R Primer
 
   | 10uM R Primer
 
   | 1.0
 
   | 1.0
 
   | 5.0
 
   | 5.0
 +
  | Annealing
 +
  | 66
 +
  | 30
 +
  | 35x
 
|-
 
|-
 
   | 10mM dNTPs
 
   | 10mM dNTPs
 
   | 1.0
 
   | 1.0
 
   | 5.0
 
   | 5.0
 +
  | Elongation
 +
  | 72
 +
  | 60
 +
  | 35x
 
|-
 
|-
 
   | Phusion Pol.
 
   | Phusion Pol.
 
   | 0.5  
 
   | 0.5  
 
   | 2.5
 
   | 2.5
 +
  | Elongation
 +
  | 72
 +
  | 600
 +
  | 1x
 
|-
 
|-
 
   | 5x HF buffer
 
   | 5x HF buffer
 
   | 10
 
   | 10
 
   | 50
 
   | 50
 +
  | Cold Storage
 +
  | 4
 +
  | ∞
 +
  | 1x
 
|-
 
|-
 
   | dH2O
 
   | dH2O

Revision as of 19:47, 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