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

 
 
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Line 6: Line 6:
  
  
−
===Design Notes===
+
==Design Notes==
−
Whatever
+
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 [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
 +
  |+ 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:
 +
 +
{| border=1
 +
  |+ 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.
 +
 +
<div style="text-align: center;">Gel Verification of Ligation of AubR-MRV</div>
 +
 +
<div style="text-align: center;">[[File:T--Arizona_State--Gel1.jpg]]</div>
 +
<div style="text-align: center;">Columns A and B are for the AubR sequence. The first lane contains the 1kb+ DNA ladder. </div>
 +
===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:
 +
 +
{| border=1
 +
  |+ Promoter Ligation 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.
 +
 +
<div style="text-align: center;">Promoter Ligation Gel Verification</div>
 +
 +
<div style="text-align: center;">[[File:T--Arizona_State--Gel2.jpg]]</div>
 +
 +
<div style="text-align: center;">Column 1 is the AubR sequence in MRV. The first lane contains the 1kb+ DNA ladder.
 +
</div>
 +
 +
The expected size of pAubR is 152 bp. This gel shows that pAubR was indeed the correct length and was to be inputted into the vector.
 +
 +
===Stage 3-Create Viable Stock of AubR===
 +
The completion of the AubR part was completed by Rene Davis and Jiaqi Wu. A simple transformation was done into E. Coli BL21 strain, which was grown overnight and compared against a negative control. The AubR plate produced an ample amount of colonies (~50) while the negative control did not produce any. Colonies from this plate were picked and run under gel verification, which produced the image below:
 +
 +
<div style="text-align: center;">Gel Verification of Complete AubR Sequence</div>
 +
 +
<div style="text-align: center;">[[File:T--Arizona_State--Gel4.jpg]]</div>
 +
 +
<div style="text-align: center;">Lanes 7-9 contain AubR samples, while Lane 1 contains the 1kb+ ladder</div>
 +
 +
With the confirmation from the gel verification (and further sequencing), the AubR part was successfully produced, and was used thoroughly in experiments conducted for this year's project.
 
===Source===
 
===Source===
  
−
Whatever
+
This HSL receiver was recovered from a metagenomic project on soil. The specific source is unknown.
  
 
===References===
 
===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

Latest revision as of 07:54, 19 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
Columns A and B are for the AubR sequence. The first lane contains the 1kb+ DNA ladder.

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:

Promoter Ligation 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.

Promoter Ligation Gel Verification
T--Arizona State--Gel2.jpg
Column 1 is the AubR sequence in MRV. The first lane contains the 1kb+ DNA ladder.

The expected size of pAubR is 152 bp. This gel shows that pAubR was indeed the correct length and was to be inputted into the vector.

Stage 3-Create Viable Stock of AubR

The completion of the AubR part was completed by Rene Davis and Jiaqi Wu. A simple transformation was done into E. Coli BL21 strain, which was grown overnight and compared against a negative control. The AubR plate produced an ample amount of colonies (~50) while the negative control did not produce any. Colonies from this plate were picked and run under gel verification, which produced the image below:

Gel Verification of Complete AubR Sequence
T--Arizona State--Gel4.jpg
Lanes 7-9 contain AubR samples, while Lane 1 contains the 1kb+ ladder

With the confirmation from the gel verification (and further sequencing), the AubR part was successfully produced, and was used thoroughly in experiments conducted for this year's project.

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