Difference between revisions of "Part:BBa K2033001:Design"
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====Digestion and Ligation==== | ====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: | 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: | ||
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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. | ||
Revision as of 08:11, 18 September 2016
C(12)-HSL Receiver Device - AubR
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 395
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 395
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 395
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 395
Illegal AgeI site found at 205 - 1000INCOMPATIBLE 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.
| 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:
| 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
The AubR promoter sequences were ordered as g-blocks and digested with E/X and then de-phosphorylated. 20uL of digested DNA sequence was reacted with 1uL of phosphatase to remove phosphates from the sequence. The reaction was incubated at 37°C for 10 minutes and 75°C for 2 minutes to provide sufficient heat energy for the enzyme to work. The g-block promoter sequence was ligated to the AubR sequence via the following reaction:
| Part | Volume(uL) |
|---|---|
| Insert DNA (promoter) | 5.0 |
| Vector DNA | 0.2 |
| 10x FD Buffer | 2.0 |
| 10mM DTT | 1.0 |
| 10mM ATP | 1.0 |
| FD Bbsi/Bpil | 1.0 |
| T4 DNA Ligase | 1.0 |
| dH2O | 8.8 |
| Total | 20 |
The reaction mixture was first incubated at 37°C for 5 minutes and 23°C for 5 minutes. The reactions were then mini-prepped and gel verified using EcoRI and SpeI. After three trials, the following gel was produced.
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