Difference between revisions of "Part:BBa K2033001:Design"
Line 125: Line 125:
  
 
The ligation reaction was transformed with DH5α-turbo cells and plated for overnight growth. The Ampicillin plate produced 11 colonies of AubR, of which 2 were picked for further growth in liquid cultures. After mini-prepping the liquid cultures, a gel verification was performed. The two expected bands for the AubR-MRV vector complex should be around 3200bp and 860bp. The gel shown below indicates that the ligation was successful.  
 
The ligation reaction was transformed with DH5α-turbo cells and plated for overnight growth. The Ampicillin plate produced 11 colonies of AubR, of which 2 were picked for further growth in liquid cultures. After mini-prepping the liquid cultures, a gel verification was performed. The two expected bands for the AubR-MRV vector complex should be around 3200bp and 860bp. The gel shown below indicates that the ligation was successful.  
 +
 
Gel Verification of Ligation of AubR-MRV
 
Gel Verification of Ligation of AubR-MRV
 +
 
[[File:T--Arizona State--Gel1.jpg]]   
 
[[File:T--Arizona State--Gel1.jpg]]   
 
===Stage 2-Insert Promoters===
 
===Stage 2-Insert Promoters===

Revision as of 09:39, 15 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

Digestion and Ligation

The PCR product and the cloning vector were digested with EcoRI and XbaI. These were gel verified and then combined in a 15uL ligation reaction (10 minutes at 37°C heat block). The reaction mixture is shown below:

Ligation Reaction
Reagent Reaction Volume(uL) Negative Ctrl. Volume(uL)
Insert DNA 3.0 0.0
Vector DNA 3.5 3.5
2x Ign Buffer(Roche) 7.5 7.5
T4 Ligase(NEB) 1.0 5.0
dH2O 0.0 3.0
Total 15 15

The ligation reaction was transformed with DH5α-turbo cells and plated for overnight growth. The Ampicillin plate produced 11 colonies of AubR, of which 2 were picked for further growth in liquid cultures. After mini-prepping the liquid cultures, a gel verification was performed. The two expected bands for the AubR-MRV vector complex should be around 3200bp and 860bp. The gel shown below indicates that the ligation was successful.

Gel Verification of Ligation of AubR-MRV

T--Arizona State--Gel1.jpg

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