iGEM 2018 — Team British_Columbia

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# Team:British Columbia/Notebook

[![Logo](https://static.igem.org/mediawiki/2018/4/4b/T--British_Columbia--logo.png)](https://2018.igem.org/Team%3ABritish_Columbia)

* PROJECT

  [OVERVIEW](https://2018.igem.org/Team%3ABritish_Columbia/Description)
  [DISTRIBUTED BIOSYNTHESIS](https://2018.igem.org/Team%3ABritish_Columbia/Naringenin)
  [BIOSENSOR](https://2018.igem.org/Team%3ABritish_Columbia/Biosensor)
  [POPULATION DYNAMICS](https://2018.igem.org/Team%3ABritish_Columbia/Kaempferol)
  [PARTS](https://2018.igem.org/Team%3ABritish_Columbia/Parts)
* [MODEL](https://2018.igem.org/Team%3ABritish_Columbia/Model)
* HUMAN PRACTICES

  [HUMAN PRACTICES](https://2018.igem.org/Team%3ABritish_Columbia/Human_Practices)
  [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ABritish_Columbia/Public_Engagement)
  [COLLABORATIONS](https://2018.igem.org/Team%3ABritish_Columbia/Collaborations)
* TEAM

  [MEMBERS](https://2018.igem.org/Team%3ABritish_Columbia/Team)
  [ATTRIBUTIONS](https://2018.igem.org/Team%3ABritish_Columbia/Attributions)
* LABORATORY

  [NOTEBOOK](https://2018.igem.org/Team%3ABritish_Columbia/Notebook)
  [PROTOCOLS](https://2018.igem.org/Team%3ABritish_Columbia/Protocols)
  [INTERLAB](https://2018.igem.org/Team%3ABritish_Columbia/InterLab)
* [ACHIEVEMENTS](https://2018.igem.org/Team%3ABritish_Columbia/Deliverables)

![](https://static.igem.org/mediawiki/2018/a/a5/T--British_Columbia--NB-Hdg.png)

* [Naringenin Notebook](#naringenin)
* [Biosensor Notebook](#biosensor)
* [Kaempferol Notebook](#kaempferol)

### Naringenin Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:

Constitutive promoter + RBS
BBa\_K880005
~70 BP
Spring 2018 Distribution
Plate 2, Well 3F
pSB1C3 (Cm)

BBa\_K880005
~70 BP
Spring 2018 Distribution
Plate 2, Well 3F
pSB1C3 (Cm)

4CL
BBa\_K801093
~1700 BP
Spring 2018 Distribution
Plate 2, Well 17D
pSB1C3 (Cm)

RBS+TAL
BBa\_K1033000
~1600
Spring 2018 Distribution
Plate 4, Well 6C
pSB1C3 (Cm)

First, TAL and 4CL with RBS (ribosomal binding site) to combine them:

* 4CL digest with EcoRI + SpeI
* TAL digest with EcoRI + Xbal
* Ligation of both digestion with T4 ligase
* E. coli transformation via heat shock method
* Spread on CMB-LB agar plate and incubate at 37 C° overnight
* PCR and gel electrophoresis to analyze if successful
* Take positive colonies and place in LB with CMP incubate for 16 hours at 37\*C
* Plasmid obtained

Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):

* CHS digest with EcoRI and SpeI
* CHI digest with EcoRI and Xbal
* Ligation of both digestion with T4 ligase
* E. coli transformation via heat shock method
* Spread on CMB-LB agar plate at 37c° overnight
* PCR and gel electrophoresis to analyze if successful
* Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
* Plasmid obtained

Third, combine the two plasmids previously prepared:

* 4CL – TAL digest by EcoRI and SpeI
* CHS-CHI with EcoRI and Xbal
* Dephosphorylate CHS-CHI
* Ligate the two together with T4 ligation
* Add GFP via digestion and then ligation\*
* E. coli transformation via heat shock method
* PCR and gel electrophoresis to analyze if successful
* Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

Protocol for cloning promoter-RBS-4CL in pSB1C3

A. Restriction enzyme digest
1. Prepare the master mix in a microfuge tube by combining the following:
Component
Volume to add (ul)
10X NEB Buffer 2.1
6
NEB PstI restriction enzyme
4
Autoclaved distilled water
26
TOTAL
36
2. Zip-spin the master mix to collect all liquid at the bottom of the tube
3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix
4. Vortex the master mix thoroughly
5. Zip-spin the master mix again
6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix)
7. Label the three tubes containing the aliquots as follows:
a. P-RBS
b. 4CL
c. PDC (positive digest control)

### Biosensor Notebook

### July

* 26:
  + Transformed RFP + RBS into DH5a
  + Transformed GFP + RBS into DH5a
* 27:
  + Inoculated RFP + RBS in 25mg/uL of CM-25
  + Inoculated GFP + RBS in 25mg/uL of CM-25
* 28:
  + Miniprepped RFP + RBS
  + Miniprepped GFP + RBS
  + Digested RFP + RBS with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL
  + Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL
  + Eventually discarded both digests as we did not use them

---

### August

* 7:
  + Transformed Bba\_E0020 into DH5a
* 8:
  + Inoculated CFP strains with 25mg/uL of CM-25
* 9:
  + Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL
* 15:
  + Transformed RFP into DH5a
* 16:
  + Inoculated RFP in 25mg/uL of CM-25
  + Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL
* 18:
  + Transformed Bba\_E0040 (GFP) into DH5a
  + Transformed Bba\_l15016 (CFP + RBS) into DH5a
  + Synthesized parts from IDT were resuspended in 1X TBE
  - FdeR
  - Pt181-repressor
  - Pt181-GP2
  - GP2
  - Antisense+ RFP, RFP+RBS, CFP, GFP+RBS have all been digested
* 19:
  + Transformed Bba\_E0040 (GFP) into DH5a
  + Transfroemd CFP + RBS into DH5a
  + Both transformations were unsuccessful
* 20
  + OD of DH5a competent cells was 0.69
  + Pelleted competent cells using 0.1M CaCl2
  + Transformed Bba\_E0040 (GFP) into DH5a
  + Transformed Bba\_I5016 (CFP + RBS) into DH5a
* 21:
  + OD of DH5a competent cells was 0.53
  + Pelleted competent cells using 0.1M CaCl2
  + Positive and Negative controls did not function properly
  + Realized glycerol was not added
  + Re-setup experiment
* 23:
  + Reaction cleanup using ABM kit, involving:
  - GP2
  - E+S
  - Pt181-GP2
  - FdeR+ Transformed Bba\_E0040 (GFP) into DH5a
  + Transformed Bba\_I15016 (CFP + RBS) into DH5a
  + Both transformations were unsuccessful
* 24
  + Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer
  + Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer
  + Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer
  + Purified with ABM kit:
  - X+P
  - E+P
  - E+S
  - CFP
  - GFP+RBS
  - RFP+RBS
  - RFP+ Inoculated CFP and GFP in 25mg/uL of CM-25
* 25:
  + Miniprepped CFP+RBS and GFP (concentration 149ng/uL)* 27:
    + Ligated FdeR and Repressor DNA into RFP vector
    + Ligated FdER and Repressor DNA into E+S vector
    + Ligated pt181-GP2 and GP2 DNA into E+P
    + Ligated antisense DNA into X+P
  * 29:
    + Transformed in DH5a:
      RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  * 30:
    + Inoculated in 25mg/uL of CM25:
      RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  * 31:
    + Miniprepped:
      RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense

---

### September

- 4:
  * Digested:
    RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- 5:
  * Ran Gel Electrophoresis In 1X TBE buffer on:
    RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- 7:
  * Double checked digestions with BamH1 + ECOR1 + Pst1 of strains:
    RFP - FdeR, E+S-FdeR
- 13:
  * Transformed RFP - FdeR2 into DHd5a
  * Digested GFP Vector using: E+P, E+X, E+S
- 20:
  * Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)
- 22:
  * Purified: GFP - E+P, GFP - E+S
  * Digested Repressor (from IDT) using E+S
  * Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector
- 23:
  * Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- 24:
  * Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- 24:
  * Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- 25:
  * Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig

---

### Kaempferol Notebook

**We kindly received the following from UBC iGEM distribution kit of 2018:**

Constitutive promoter + RBS
BBa\_K880005
~70 BP
Spring 2018 Distribution
Plate 2, Well 3F
pSB1C3 (Cm)

F3H
BBa\_K1497009
~1107 BP
Spring 2018 Distribution
Plate 2, Well 12I
pSB1C3 (Cm)

RBS
BBa\_B0034
~12 BP
Spring 2018 Distribution
Plate 4, Well 1N
pSB1C3 (Cm)

Double Terminator
BBa\_B0015
~129 BP
Spring 2018 Distribution
Plate 3, Well 3F
pSB1C3 (Cm)

**Parts we've synthesized ourselves:**

FLS1
BBa\_K2700000
~1011 BP
pSB1C3 (Cm)

### July

* 25:
  + Transformed F3H into DH5a
  + Transformed K880005 into DH5a
  + Transformed B0034 into DH5a
  + Transformed B0015 into DH5a
* 26:
  + Inoculated F3H in 25mg/uL of CM-25
  + Inoculated K880005 in 25mg/uL of CM-25
  + Inoculated B0034 in 25mg/uL of CM-25
  + Inoculated B0015 in 25mg/uL of CM-25
* 27:
  + Miniprepped F3H
  + Miniprepped K880005
  + Miniprepped B0034
  + Miniprepped B0015

- 28:
  * Digested F3H with XbaI and SpeI
  * Digested K880005 with SpeI and Pst1
  * Digested B0034 with EcoR1 and Xba1
  * Digested B0015 with EcoR1 and Xba1

---

### August

* 2:
  + Ran gel and purified all samples+ Ligate F3H (insert, 5uL) and K880005 (vector, 2uL) with T4 ligase
* 7:
  + transformed F3H and K880005
  + Digested RFP in pSB1C3; ran gel and purified
  + Digested synthesized FLS1 with Ecor1 and Pst1
* 10:
  + inoculated F3H and K880005
* 15:
  + Miniprepped F3H + K880005
  + Gel ran and get purified for RFP/pSB1C3, F3H + K880005, B0034, B0015
  + gel ran and PCR purified for FLS1
* 16:
  + Redigested F3H with XbaI and SpeI (gel failed on the 15th, needed to redo cloning)
  + Redigested K880005 with SpeI and Pst1
* 17:
  + Ligated RFP/pSB1C3 (vector, 2uL) with FLS1 (insert, 4uL)
  + Ligated FLS1 (vector, 2uL) with B0015 (insert, 4uL)
  + purified and religated F3H+K880005
* 19:
  + Transformed FLS1 + pSB1C3 into DH5a
  + Transformed FLS1 + B0015 into DH5a
  + Transformed F3H + K880005 into DH5a
* 20
  + Inoculate all samples
* 21:
  + Miniprep all samples
* 24
  + Ran diagnostic gel for all samples, F3H + K880005 failed again, everything else fine
* 25:
  + Took originally miniprepped F3H and digested with XbaI and SpeI* 27:
    + Gel purified F3H
    + ligated with previously digested B0034

  ---

  ### September

  * 5:
    + Transformed F3H + B0034
  * 5:
    + Inoculated F3H + B0034
  * 7:
    + Miniprepped F3H + B0034
  * 10:
    + Ran F3H + B0034 on gel and gel purified (cloning successful!)
  * 15:
    + Used glycerol stock K880005 and streaked on LB+Cm plate
  * 16:
    + Inoculated K880005
  * 17:
    + Miniprepped K880005
  * 19:
    + Digested K880005 with Spe1 + Pst1
  * 20:
    + Ran K880005 in gel and gel purified
  * 23:
    + Digested FLS1 + B0015 with Pst1 + Spe1
    + Digested F3H + B0034 with Xba1 + Pst1
  * 24:
    + Gel eletrophoresis and gel purified FLS1 + B0015
    + Gel eletrophoresis and gel purified F3H + B0034
  * 29:
    + Ligation of F3H + B0034 (insert, 6uL) and K880005 (vector, 2uL)

  ---

  ### October

  * 1:
    + Transformation of k880005 + F3H + B0034
  * 2:
    + Inoculation of k880005 + F3H + B0034
  * 3:
    + Miniprep of k880005 + F3H + B0034
  * 4:
    + Digest of k880005 + F3H + B0034

### Plasmid Maintenance Notebook


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# Team:British Columbia/Experiments

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[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3ABritish_Columbia)
[HOME](https://2018.igem.org/Team%3ABritish_Columbia)

TEAM

[Team Members](https://2018.igem.org/Team%3ABritish_Columbia/Team)
[Collaborations](https://2018.igem.org/Team%3ABritish_Columbia/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ABritish_Columbia/Description)
[Design](https://2018.igem.org/Team%3ABritish_Columbia/Design)
[Experiments](https://2018.igem.org/Team%3ABritish_Columbia/Experiments)
[Notebook](https://2018.igem.org/Team%3ABritish_Columbia/Notebook)
[InterLab](https://2018.igem.org/Team%3ABritish_Columbia/InterLab)
[Model](https://2018.igem.org/Team%3ABritish_Columbia/Model)
[Results](https://2018.igem.org/Team%3ABritish_Columbia/Results)
[Demonstrate](https://2018.igem.org/Team%3ABritish_Columbia/Demonstrate)
[Improve](https://2018.igem.org/Team%3ABritish_Columbia/Improve)
[Attributions](https://2018.igem.org/Team%3ABritish_Columbia/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ABritish_Columbia/Parts)
[Basic Parts](https://2018.igem.org/Team%3ABritish_Columbia/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ABritish_Columbia/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ABritish_Columbia/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ABritish_Columbia/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ABritish_Columbia/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ABritish_Columbia/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ABritish_Columbia/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ABritish_Columbia/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ABritish_Columbia/Hardware)
[Measurement](https://2018.igem.org/Team%3ABritish_Columbia/Measurement)
[Model](https://2018.igem.org/Team%3ABritish_Columbia/Model)
[Plant](https://2018.igem.org/Team%3ABritish_Columbia/Plant)
[Software](https://2018.igem.org/Team%3ABritish_Columbia/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=British_Columbia)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:British Columbia/Results

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[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3ABritish_Columbia)
[HOME](https://2018.igem.org/Team%3ABritish_Columbia)

TEAM

[Team Members](https://2018.igem.org/Team%3ABritish_Columbia/Team)
[Collaborations](https://2018.igem.org/Team%3ABritish_Columbia/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ABritish_Columbia/Description)
[Design](https://2018.igem.org/Team%3ABritish_Columbia/Design)
[Experiments](https://2018.igem.org/Team%3ABritish_Columbia/Experiments)
[Notebook](https://2018.igem.org/Team%3ABritish_Columbia/Notebook)
[InterLab](https://2018.igem.org/Team%3ABritish_Columbia/InterLab)
[Model](https://2018.igem.org/Team%3ABritish_Columbia/Model)
[Results](https://2018.igem.org/Team%3ABritish_Columbia/Results)
[Demonstrate](https://2018.igem.org/Team%3ABritish_Columbia/Demonstrate)
[Improve](https://2018.igem.org/Team%3ABritish_Columbia/Improve)
[Attributions](https://2018.igem.org/Team%3ABritish_Columbia/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ABritish_Columbia/Parts)
[Basic Parts](https://2018.igem.org/Team%3ABritish_Columbia/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ABritish_Columbia/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ABritish_Columbia/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ABritish_Columbia/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ABritish_Columbia/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ABritish_Columbia/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ABritish_Columbia/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ABritish_Columbia/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ABritish_Columbia/Hardware)
[Measurement](https://2018.igem.org/Team%3ABritish_Columbia/Measurement)
[Model](https://2018.igem.org/Team%3ABritish_Columbia/Model)
[Plant](https://2018.igem.org/Team%3ABritish_Columbia/Plant)
[Software](https://2018.igem.org/Team%3ABritish_Columbia/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=British_Columbia)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)