Difference between revisions of "Part:BBa K2033001:Design"

Revision as of 09:38, 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.

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

@@ Line 124: / Line 124: @@
 |}
-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 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.
+[[File:T--Arizona State--Gel1.jpg]]
 ===Stage 2-Insert Promoters===