iGEM 2019 — Team UCopenhagen

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UCopenhagen/Notebook

* [![](https://static.igem.org/mediawiki/2019/0/0b/T--UCopenhagen--Onlytextlogo.png)](https://2019.igem.org/Team%3AUCopenhagen)
* Project
  + [Description](https://2019.igem.org/Team%3AUCopenhagen/Description)
  + [Design](https://2019.igem.org/Team%3AUCopenhagen/Design)
  + [Results](https://2019.igem.org/Team%3AUCopenhagen/Results)
  + [Future Plans](https://2019.igem.org/Team%3AUCopenhagen/FuturePlans)
* Lab
  + [Parts Overview](https://2019.igem.org/Team%3AUCopenhagen/Parts)
  + [Protocols](https://2019.igem.org/Team%3AUCopenhagen/Experiments)
  + [Safety](https://2019.igem.org/Team%3AUCopenhagen/Safety)
  + [Notebook](https://2019.igem.org/Team%3AUCopenhagen/Notebook)
  + [Modeling](https://2019.igem.org/Team%3AUCopenhagen/Model)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AUCopenhagen/Human_Practices)
  + [Entrepreneurship](https://2019.igem.org/Team%3AUCopenhagen/Entrepreneurship)
  + [App](https://2019.igem.org/Team%3AUCopenhagen/App_Description)
  + [Education and Engagement](https://2019.igem.org/Team%3AUCopenhagen/Public_Engagement)
* Collaboration
  + [Collaborations](https://2019.igem.org/Team%3AUCopenhagen/Collaborations)
  + [Meetups](https://2019.igem.org/Team%3AUCopenhagen/Meetups)
  + [Nordic iGEM Conference](https://2019.igem.org/Team%3AUCopenhagen/NiC)
* Team
  + [Team Members](https://2019.igem.org/Team%3AUCopenhagen/Team)
  + [Team Building](https://2019.igem.org/Team%3AUCopenhagen/Team_Building)
* Acknowledgements
  + [Attributions](https://2019.igem.org/Team%3AUCopenhagen/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AUCopenhagen/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/a/a6/T--UCopenhagen--Logo_Awards.png)

  + [Judging form](https://igem.org/2019_Judging_Form?team=UCopenhagen)
  + [Medal Requirements](https://2019.igem.org/Team%3AUCopenhagen/Medals)
  + [Awards](https://2019.igem.org/Team%3AUCopenhagen/Awards)

![](https://static.igem.org/mediawiki/2019/2/2b/T--UCopenhagen--Notebookbckgr2.jpg)
![](https://static.igem.org/mediawiki/2019/7/7c/T--UCopenhagen--Almostblank.png)

To scrutinize the results and properly understand the day to day progress of the Ovulaid team’s scientific work an exhaustive online notebook was kept, completely reflecting the team’s physical laboratory notebooks. The online notebook is structured by week and underneath you can see the experiments carried out that week. To see the details of each day and experiment, click on the week number.

## Biosensor Notebook

* [Week 21 (20th-26th of May)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_21)
  + CONSTRUCT AND PRIMER DESIGN
  + DILUTION OF PRIMERS AND GENE FRAGMENTS
  + NORMAL PCR AMPLIFICATION: X7-PFU POLYMERASE
  + GEL ELECTROPHORESIS
  + PURIFICATION OF PCR PRODUCT
* [Week 22 (27th of May-2nd of June)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_22)
  + NANODROP
  + ADAPTED USER REACTION (LIGATION)
  + MAKING PLATES FOR E.COLI TO GROW
  + E.COLI TRANSFORMATION
  + COLONY PCR (E.COLI)
  + GEL ELECTROPHORESIS
  + TRANSFORMED E. COLI COLONIES INTO LIQUID CULTURE
  + PLASMID DNA EXTRACTION
* [Week 23 (3rd-9th of June)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_23)
  + SEQUENCING
  + USER-LIGATION/E.COLI TRANSFORMATION PART 2: SAMPLE 4 & 3
  + COLONY PCR
  + GEL ELECTROPHORESIS
* [Week 24 (10th-16th of June)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_24)
  + DNA EXTRACTION
  + SEQUENCING OF SAMPLE S3C4 AND S3C5
  + ADAPTED USER LIGATION SAMPLE 1, 2, 4, 6
* [Week 25 (17th-23rd of June)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_25)
  + EXAM WEEK
* [Week 26 (24th-30th of June)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_26)
  + COMPETENT CELLS
  + NORMAL PCR AMPLIFICATION: X7-PFU POLYMERASE
  + GEL ELECTROPHORESIS FOR SAMPLE 1, 11, 2 AND 12
  + DNA PURIFICATION FOR SAMPLE 1, 2, 11 AND 12
  + E. COLI TRANSFORMATION SAMPLES 1, 2, 4, 6
  + MAKING OUR OWN USER VECTOR STOCK
  + COLONY PCR FOR SAMPLES 1, 2, 4 AND 6
  + COMPETENT CELL TEST
  + GEL ELECTROPHORESIS FOR SAMPLES 1, 2, 4, AND 6
  + PLASMID PURIFICATION
* [Week 27 (1st-7th of July)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_27)
  + PCR AMPLIFICATION SAMPLE 1,2, 11 AND 12+ GEL ELECTROPHORESIS FOR SAMPLE 1, 2, 11 AND 12+ PLASMID DNA EXTRACTION+ E.COLI INCUBATION FOR VECTOR BACKBONE AMPLIFICATION+ PREPARING SAMPLES FOR SEQUENCING+ COMPETENT CELL TEST+ PLASMID PURIFICATION+ GEL ELECTROPHORESIS+ FIRST HUMAN LHCGR PCR AMPLIFICATION: X7-PFU POLYMERASE+ GEL ELECTROPHORESIS
* [Week 28 (8th-14th of July)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_28)
  + 2nd PCR AMPLIFICATION: X7-PFU POLYMERASE AND GEL ELECTROPHORESIS
  + SEQUENCING FOR GPER, GPER-sfGFP, XLHCGR and XLHCGR-sfGFP
  + 3RD PCR AMPLIFICATION: X7-PFU POLYMERASE AND GEL ELECTROPHORESIS
  + 4th PCR AMPLIFICATION: X7-PFU POLYMERASE AND GEL ELECTROPHORESIS
  + YEAST TRANSFORMATION
  + PCR AMPLIFICATION
* [Week 29 (15th-21st of July)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_29)
  + COLONY PCR OF TRANSFORMED YEAST+ REDO OF YEAST COLONY PCR+ O/N CULTURES FOR GLYCEROL STOCKS+ GLYCEROL STOCKS+ PLASMID PURIFICATION+ PCR AMPLIFICATION
* [Week 30 (22nd-28th of July)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_30)
  + YEAST TRANSFORMATION+ MEDIA AND REAGENT PREPARATION+ PRIMER DILUTION+ EZNA PLASMID PURIFICATION+ SAMPLE PREPARATION FOR SEQUENCING+ GLYCEROL STOCK+ EZNA PLASMID PURIFICATION+ GEL ELECTROPHORESIS OF OLD PCR PRODUCTS+ E. COLI TRANSFORMATION WITH Ass2A-pPGK1-GPA1-Gas+ COLONY PCR I OF TRANSFORMED YEAST FROM 22ND JULY 2019
* [Week 31 (29th of July-4th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_31)
  + GEL ELECTROPHORESIS OF E.COLI TRANSFORMATION CONSTRUCTS AND YEAST COLONY PCR SAMPLE+ COLONY PCR OF TRANSFORMED E. COLI+ COLONY PCR II OF TRANSFORMED YEAST FROM 22ND JULY 2019+ INOCULATION OF POSITIVE YEAST COLONIES OF CONSTRUCT X3A-pCCW12-GPER-sfGFP+ PLASMID PURIFICATION FOR Ass2A-pPGK1-GPA1-Gas EXPRESSING E. COLI+ FLUORESCENCE MICROSCOPE OF POSITIVE YEAST COLONIES+ PCR AMPLIFICATION of TPST1 & TPST2+ YEAST TRANSFORMATION
* [Week 32 (5th-11th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_32)
  + EZNA PLASMID PURIFICATION+ YEAST COLONY PCR+ COLONY PCR OF YEAST+ INOCULATION FROM PLATES+ USER LIGATION+ GEL ELECTROPHORESIS+ YEAST STREAKING+ GLYCEROL STOCKS+ PREPARATION OF OLD SAMPLES FOR SEQUENCING
* [Week 33 (12th-18th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_33)
  + USER LIGATION+ E. COLI TRANSFORMATION+ REPETITION OF USER LIGATION+ REPETITION OF E. COLI TRANSFORMATION
* [Week 34 (19th-25th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_34)
  + REPETITION OF COLONY PCR+ USER LIGATION
* [Week 35 (26th of August-1st of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_35)
  + YEAST COLONY PCR+ GLU-U AGAR PREPARATION+ RUN OF GEL WITH COLONY PCR SAMPLE FROM 28/8+ PLASMID LIGATION+ INCUBATION OF YEAST CULTURES+ YEAST TRANSFORMATION+ GLYCEROL STOCKS
* [Week 36 (2nd-8th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_36)
  + YEAST COLONY PCR+ SETTING UP E.COLI CULTURES+ PROTEIN PLASMID PURIFICATION OF E.COLI CULTURES+ NANODROP
* [Week 37 (9th-15th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_37)
  + SETTING-UP CULTURES FOR BIOACTIVITY ASSAY+ FIRST ESTRADIOL BIOACTIVITY ASSAY
* [Week 38 (16th-22nd of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_38)
  + SECOND ESTRADIOL BIOACTIVITY ASSAY
* [Week 39 (23rd-29th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_39)
  + Third Estradiol bioactivity assay
  + Fluorescence Microscopy
* [Week 40 (30th of September-6th October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_40)
  + Fluorescence Confocal Microscopy
  + Miniprep - Plasmid DNA purification
* [Week 41 (7th-13th October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_41)
  + Bioactivity assay
* [Week 42 (14th-20th October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/Week_42)
  + 1st Western Blot with anti-GFP antibodies
  + Confocal microscopy
  + Repetition of the anti-sfGFP Western Blot

## Kill Switch Notebook

* [Week 32 (5th-11th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week32)
  + PRIMERS AND G-BLOCKS DILUTION+ PCR AMPLIFICATION OF BAX AND BI-1+ GEL ELECTROPHORESIS+ PCR CLEAN UP
* [Week 33 (12th-18th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week33)
  + PCR AMPLIFICATION OF BI-1, GAL1, TEF1 AND ADH2+ GEL ELECTROPHORESIS OF PCR PRODUCTS+ PLASMID DIGESTION FOR USER LIGATION+ PCR CLEAN UP+ USER LIGATION+ E.COLI TRANSFORMATION+ COLONY PCR+ REPETITION OF COLONY PCR
* [Week 34 (19th-25th of August)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week34)
  + GEL ELECTROPHORESIS OF COLONY PCR PRODUCTS+ PLASMID PURIFICATION+ PREPARATIONS FOR YEAST TRANSFORMATION+ PLASMID DIGESTION+ YEAST TRANSFORMATION
* [Week 35 (26th of August-1st of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week35)
  + YEAST COLONY PCR+ GEL ELECTROPHORESIS FOR YEAST COLONY PCR
* [Week 36 (2nd-8th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week36)
  + SET UP A CULTURE OF THE X12C VECTOR+ GAL-INDUCTION ASSAY: MEDIA PREPARATION+ GAL-INDUCTION ASSAY: PREPARING PLATES+ GAL-INDUCTION ASSAY: PREPARE A CULTRE AND PLATE IT+ PURIFICATION OF X12C
* [Week 37 (9th-15th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week37)
  + SECOND GAL-INDUCTION ASSAY+ PLASMID DIGESTION+ SAMPLE DIGESTION FOR YEAST TRANSFORMATION+ SETTING UP YEAST FOR TRANSFORMATION
* [Week 38 (16th-22nd of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week38)
  + YEAST COLONY PCR+ YEAST COLONY PCR+ STORING OF POSTIVE pGAL1-BAX CULTURE
* [Week 39 (23rd-29th of September)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week39)
  + NO EXPERIMENTS
* [Week 40 (30th of September-6th of October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week40)
  + INOCULATION OF E.COLI CONTAINING pTEF-BI-1 & pGAL1-BI-1
* [Week 41 (7th-13th of October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week41)
  + Inoculation for gal-induction assay+ Sequencing+ Gal-induction assay (liquid) of pGAL1-BAX+ Preparing plates for gal-induction assay of the plasmid constructs+ Dual plasmid transformation+ Preparation of plates for the plasmid construct assays+ Miniprep+ Gal-induction assay of pGAL1-BAX+ Inoculation of yeast O/N cultures for WB
* [Week 42 (14th-20th of October)](https://2019.igem.org/Team%3AUCopenhagen/Notebook/killswitch/week42)
  + Restreak ADH2-BAX+GAL1-BI plasmid transformants+ Yeast transformation+ Restreaks from Monday were checked+ Gal-induction spot test of integrated and plasmid-expressed pGAL1-BAX+ Restreak of restreak was made

# About Us

We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

![](https://static.igem.org/mediawiki/2019/6/61/T--UCopenhagen--LgogForBlack.png)

# Keep in Touch

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iGEM Team Copenhagen

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iGEM\_Copenhagen

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iGEM\_Copenhagen

![](https://static.igem.org/mediawiki/2019/a/a2/T--UCopenhagen--Emaillogo.png)
UCPH.IGEM2019@gmail.com

# Address

University of Copenhagen

Thorvaldsensvej 40, Frederiksberg C

Denmark

![](https://static.igem.org/mediawiki/2019/7/72/T--UCopenhagen--KUlogo.png)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UCopenhagen/Experiments

* [![](https://static.igem.org/mediawiki/2019/0/0b/T--UCopenhagen--Onlytextlogo.png)](https://2019.igem.org/Team%3AUCopenhagen)
* Project
  + [Description](https://2019.igem.org/Team%3AUCopenhagen/Description)
  + [Design](https://2019.igem.org/Team%3AUCopenhagen/Design)
  + [Results](https://2019.igem.org/Team%3AUCopenhagen/Results)
  + [Future Plans](https://2019.igem.org/Team%3AUCopenhagen/FuturePlans)
* Lab
  + [Parts Overview](https://2019.igem.org/Team%3AUCopenhagen/Parts)
  + [Protocols](https://2019.igem.org/Team%3AUCopenhagen/Experiments)
  + [Safety](https://2019.igem.org/Team%3AUCopenhagen/Safety)
  + [Notebook](https://2019.igem.org/Team%3AUCopenhagen/Notebook)
  + [Modeling](https://2019.igem.org/Team%3AUCopenhagen/Model)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AUCopenhagen/Human_Practices)
  + [Entrepreneurship](https://2019.igem.org/Team%3AUCopenhagen/Entrepreneurship)
  + [App](https://2019.igem.org/Team%3AUCopenhagen/App_Description)
  + [Education and Engagement](https://2019.igem.org/Team%3AUCopenhagen/Public_Engagement)
* Collaboration
  + [Collaborations](https://2019.igem.org/Team%3AUCopenhagen/Collaborations)
  + [Meetups](https://2019.igem.org/Team%3AUCopenhagen/Meetups)
  + [Nordic iGEM Conference](https://2019.igem.org/Team%3AUCopenhagen/NiC)
* Team
  + [Team Members](https://2019.igem.org/Team%3AUCopenhagen/Team)
  + [Team Building](https://2019.igem.org/Team%3AUCopenhagen/Team_Building)
* Acknowledgements
  + [Attributions](https://2019.igem.org/Team%3AUCopenhagen/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AUCopenhagen/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/a/a6/T--UCopenhagen--Logo_Awards.png)

  + [Judging form](https://igem.org/2019_Judging_Form?team=UCopenhagen)
  + [Medal Requirements](https://2019.igem.org/Team%3AUCopenhagen/Medals)
  + [Awards](https://2019.igem.org/Team%3AUCopenhagen/Awards)

![](https://static.igem.org/mediawiki/2019/c/ca/T--UCopenhagen--Protocolbckgr2.jpg)
![](https://static.igem.org/mediawiki/2019/7/7c/T--UCopenhagen--Almostblank.png)

To see the standard protocols used consistently in the scientific work of the UCopenhagen 2019 team, see below. Smaller adaptations have been conducted according to need but these are always described in the [online notebook](%E2%80%9Dhttps%3A//2019.igem.org/Team%3AUCopenhagen/Notebook%E2%80%9D). **Click the arrows to see our protocols.**

PCR clean up
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• PCR clean up kit (E.N.Z.A)
• PCR reaction product

**Procedure:**
1.) Transfer 30-50 μL of the PCR product to a new 1.5 mL microcentrifuge tube.
2.) Add 4-5 times volume of the CP buffer.
3.) Briefly vortex and centrifuge the samples.
4.) Insert the Hibind DNA mini columns into the 2 mL collection tubes.
5.) Add the samples from step 3 to the Hibind DNA mini column.
6.) Centrifuge at 13000 g for 60 seconds at room temperature. Discard the filtrate and re use the collection tubes.
7.) Add 700 μL of DNA wash buffer diluted with ethanol to the tubes.
8.) Centrifuge at maximum speed for 60 seconds. Discard the filtrate and re use the collection tubes.
9.) Repeat step 7 for a second DNA wash buffer step.
10.) Centrifuge the empty Hibind DNA columns at maximum speed for 2 minutes to dry the column. This step is critical to remove the trace ethanol.
11.) Transfer the Hibind DNA column to a new 1.5 mL microcentrifuge tube.
12.) Add 50 μL of the elution buffer directly into the Hibind DNA column.
13.) Keep at room temperature for 2 minutes and centrifuge at maximum speed for 60 seconds.
14.) Store the DNA at -20℃.

Plasmid DNA extraction
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• E.N.Z.A Plasmid DNA mini prep kit
• Culture

**Procedure:**
1.) Vortex the liquid cultures and use 1-5 mL for the plasmid extraction.
2.) Centrifuge at 10000 g for 1 min at room temperature. Discard the pellet.
3.) Add 250 μL of solution I, vortex the samples and transfer the suspension to a new microcentrifuge tube.
4.) Add 250 μL of solution II, invert the samples and wait for 2-3 minutes.
5.) Add 350 μL Solution III and invert the tube several times.
6.) Centrifuge the samples at maximum speed for 10 minutes.
7.) Place the Hibind DNA columns onto the 2 mL collection tubes.
8.) Transfer the supernatant from step 6 into the Hibind DNA columns.
9.) Centrifuge at maximum speed for 1 minute and discarded the filtrate.
10.) Add 500 μL of HBC buffer and centrifuge it at maximum speed for 1 minute and discard the supernatant.
11.) Add 700 μL DNA Wash Buffer and centrifuge it at maximum speed for 30 seconds and discard the supernatant.
12.) Repeat step 11 for a second DNA wash step.
13.) Centrifuge the empty Hibind DNA column for 2 min at maximum speed to dry the column.This will remove trace amounts of ethanol.
14.) Transfer the Hibind DNA column into a new microcentrifuge tube.
15.) Add 50 μL of Elution Buffer, let it sit at room temperature for 2 minutes and centrifuge at maximum speed for 1 minute.
16.) Store DNA at -20℃.

Nanodrop DNA quantification
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• DNA samples
• Milli-Q water
• Nanodrop device
• Pipette

**Procedure:**
1.) Clean the measurement surface with a piece of tissue and ethanol.
2.) Use 1 μL of Milli-Q water as a blank.
3.) Clean the measurement surface with a piece of tissue.
4.) Use 1 μL of sample to measure the concentration.
NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.
5.) When done measuring, wipe the measuring surface with a tissue and Milli-Q water.

Dpn1 digestion
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Purified PCR product
• Restriction enzyme Dpn1
• Milli-Q water
• 10X cut smart buffer

**Procedure:**
1.) Prepare sample in a 0.5 mL microcentrifuge tube as follows.

| Component | Volume (μL) |
| --- | --- |
| 10X cut smart buffer | 4 |
| Purified PCR product | 30 |
| Restriction enzyme Dpn1 | 1 |
| Milli-Q | 5 |

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast Colony PCR
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• NaOH solution
• MangoMix master mix solution
• F1-primer
• F2-primer
• R1-primer
• Template: Colonies from overnight plates

**Procedure:**

**Yeast cell lysis:**
1.) Calculate the number of colonies and mark required number of PCR tubes.
2.) Add 20 μL of 20mM NaoH to all the tubes.
3.) Pick selected colonies using a pipette tip and inoculate into the tubes containing NaoH.
4.) Place the tubes in the PCR machine and run it for 10 minutes at 99℃.

**PCR reaction:**
1.) Use a commercial mastermix called MangoMix. This mastermix solution contains taq polymerase and dNTPs.
2.) Take new PCR tubes and add 5 μL of Mango master mix, 0.5 μL of each primers and 3.5 μL of water to a total volume of 9 μL.
3.) Add 1 μL of the template to each of the respective tubes.
4.) Place the tubes in the PCR machine and run a programme with following steps:

30-35 cycles for steps marked in red
96 degree celsius: 30 seconds

96 degree celsius: 30 seconds
55 degree celsius: 25 seconds
72 degree celsius: 2 minutes and 30 seconds

72 degree celsius: 5 minutes
12 degree celsius: Infinity

Bacterial Colony PCR
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

**Procedure:**
1.) Selected isolated colonies from the plate and using a sterile cocktail stick, remove a small quantity (such that it is visible on the end of the stick) of cells from the plate. This will serve as the template in the PCR mix.
2.) Label the PCR tubes with a number of g-blocks to be amplified.
3.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
4.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
5.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb)
6.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Competent Cells
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• LB agar plates
• LB liquid media
• E.coli (WT)
• Eppendorfs
• TSS buffer

**Procedure:**
1.) From the overnight culture, add 1 mL of E.coli to 99 mL of fresh LB media and grow to OD600 to 0.4-0.5.
2.) Place the eppendorfs on ice and make sure that TSS buffer is chilled in the fridge (4℃).
3.) Split the culture into different falcon tubes and incubate them on ice for 10 minutes.
4.) Centrifuge the falcon tubes for 10 minutes at 3000 RPM and 4℃ ( The centrifuge needs to be cooled down to 4℃ before use).
5.) Discard the supernatant and resuspend the pellet in TSS buffer.
6.) Make aliquots into cooled eppendorf tubes and freeze them in -80℃.

Standard PCR
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

**Procedure:**
1.) Label the PCR tubes with number of g-blocks to be amplified.
2.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
3.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
4.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb).
5.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Dilution of g-blocks and primers
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Primers
• g-blocks
• mQ water

**Procedure:**
1.) Dilute the primers and g-blocks in the tubes they came in, by making the solutions as following:
2.) Add 100 μL of Milli-Q water to the g-block fragments (1000ng) to make it 10 ng/μL .
3.) For the primers, make up to 100 µM concentrations.

DNA transformation in E.coli
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Competent cells
• Vector containing DNA

**Procedure:**
1.) Add 1% volume of the vector to competent cells.
2.) Mix by gently flicking the tube. Vortex will prevent transformation.
3.) Incubate the cells on ice for 30-60 minutes.
4.) Water bath at 42℃ for 45 seconds.
5.) Again incubate on ice for 3-5 minutes.
6.) Add 950 μL of LB and place them in shaking incubator at 37℃ for 1 hour.
7.) Centrifuge at 13000 RPM for 1 minute.
8.) Discard the supernatant slowly, leaving behind 50 μL-100 μL in the tube.
9.) Resuspend the pellet using a pipette and spread on to an appropriate selection plate and incubate overnight at 37℃.

Agarose Gel Electrophoresis
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• 1% agarose
• Gel red
• Electrophoretic chamber
• Samples to be loaded
• Molecular weight marker (DNA ladder)

**Procedure:**
1.) For a 1% (w/v) gel in TAE, weigh agarose powder for a 1% (w/v) gel.
2.) Add weighed agarose in TAE buffer (1X) and warm the solution (in a microwave) until it is completely dissolved. Let the solution cool down to hand warm temperature.
3.) Pour gel in gel tray and mix well with Gel Red. For a small gel (~ 40 mL solution) add 1 μL of Gel red; for a large gel (~80 mL) add 2 µL of Gel red. Add a comb to create wells for the samples. Allow the agarose to solidify.
4.) Transfer the gel to the electrophoresis cell, remove the combs and cover the gel in TAE buffer (1X).
5.) Prepare the electrophoresis samples by adding Nucleic Acid Loading Buffer conform the manufacturer’s instructions.
6.) Load the molecular weight marker (DNA ladder) in the first well according to manufacturer’s instructions (generally 3-5 µL) and load 5-10 µL of the dyed samples in the other wells.
7.) Connect the cables of the gel tray following the colour code and run at 80-110V for 40-60 min.

Preparation of 1000X antibiotic stock solution
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Antibiotic
• Milli-Q water
• Ethanol

**Procedure:**
1.) Weigh X grams of specific antibiotic
2.) Dissolve in 95% ethanol (Chloramphenicol) or Milli-Q water (other antibiotics).

Preparation of agar plates
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• LB agar
• Milli-Q water
• Petri dishes

**Procedure:**
1.) Dissolve X grams of LB-agar in X mL of Milli-Q.
2.) Autoclave the LB-agar solution.
3.) Cool the agar down to room temperature.
4.) Add the correct amount of the correct antibiotic. (We used 2 μL of antibiotic per mL of LB agar)
5.) Under sterile conditions, pour the LB-agar in empty Petri dishes (20-25 mL per plate).
6.) Leave the Petri dishes near the flame with the lid partially off for approximately 5 minutes so the agar can solidify.

Colony picking
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Plates with colonies
• Eppendorf tubes
• Pipette tips
• Milli-Q water

**Procedure:**
1.) Add 25 μL sterile Milli-Q to X tubes.
2.) Touch with a clean pipette tip a free colony with no other colonies attached to from your plate containing the bacteria.
3.) Stir with the pipette tip containing the bacteria in one of the tubes with Milli-Q.
4.) Point carefully with the pipette tip on a clean plate with no antibiotic resistance.
5.) Repeat this for X colonies.

Generating growth curve with plate reader
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• 96 well plate
• Specific growth media
• Inducer
• Overnight culture

**Procedure:**
1.) Add X µL of specific growth media into each well.
Use at least 2 wells for blank, add X µL media in these.
2.) Inoculate X wells per culture with X µL of the starter culture.
3.) An hour after inoculation, induce the cells with the necessary inducer (estrogen/ galactose in our case) in different concentrations as per the requirements.
4.) After induction, monitor the growth and fluorescence using a plate reader.

Restriction digestion assay
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• 10X cut smart buffer
• Restriction enzymes
• DNA sample

**Procedure:**
1.) Decide on which restriction enzyme(s) to cut with. Find out what buffer the enzyme(s) work(s) in. In general, SmartCut buffer can be used.
2.) Prepare the sample as follows:

| Component | Volume (μL) |
| --- | --- |
| DNA | (~1 µg is required) |
| Buffer (CutSmart) | 2 |
| Restriction enzyme(s) (5u/µg) | 1 µL each |
| Milli-Q | Up to 20-25 µL |

2.) Incubate for one hour at 37 °C.
3.) Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.
NOTE: This last step can be skipped if the sample is evaluated on gel immediately after.

Raffinose media preparation
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Raffinose
• Yeast nitrogen base
• Synthetic drop out media
• Amino acids
• NaOH

**Procedure:**
1.) Add all the necessary reagents in necessary quantities.
2.) The quantities used to prepare 1L of the media are given below:
Raffinose - 20g
Yeast nitrogenous base - 6.7g
Synthetic drop out media - 1.4g
Amino acids:
0.04g Trp, 0.04g Leu, 0.02 His
3.) Make up the volume to 1 L.
4.) Adjust the pH(5.8-6.2) using 10% NaOH.

USER ligation
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

This experiment is carried out to ligate the gene of interest with its promoter and vector backbone.
Note: The Gene of interests used in this experiment have already been amplified with primers containing USER sequence.
**Reagents required:**
• USER enzyme
• Cut smart buffer
• Promoter
• Gene of interest
• Vector backbone
• Milli-Q water

**Procedure:**
1.) Create a master mix using Milli-Q water, USER enzyme and cutsmart buffer as shown below.

2.) For each ligation, add the gene fragments, promoters and vector backbones separately.
3.) Add 7 μL of the master mix to each tube. Finally, place the samples in the PCR and set the following conditions:
37 °C for 30 min
25 °C for 15 min
20 °C for 10 min
12 °C indefinitely

| Compound | Volume (μL) |
| --- | --- |
| USER enzyme | 1 |
| CutSmart buffer | 1 |
| Promoter | 1 |
| Gene | 1 |
| Vector backbone | 1 |
| Vector backbone | 1 |
| Milli-Q | 5 |
| Total | 10 |

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast transformation
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• O/N yeast culture
• YPD media
• Cuvettes
• Salmon sperm
• Lithium acetate
• Vector backbones
• Milli-Q

**Procedure:**
1.) Mix 0.9 mL YPD media and 0.1 mL of overnight culture in a cuvette and measure OD at 600nm.

2.) Dilute the culture to an OD(600nm) = 0.25 based on the OD value measured.
3.) Incubate the culture for another 4h to reach an OD(600nm) of approximately 1.
4.) Boil the ssDNA(Salmon sperm) for 10 mins at 99°C in the heat block and keep it on ice.
5.) Spin down the culture in the falcon tube at 3000RCF for 10 mins at room temperature.
6.) Pour the supernatant into a waste tube and resuspend in 1mL of LiAc.
7.) Transfer the culture into 1.5 mL of Eppendorf tubes and spin down at 7000 RCF for 1 min at room temperature.
8.) Remove the supernatant carefully with a pipette and resuspend the pellet in a 100 μL of 0.1M LiAc.
9.) Add 30 μL of salmon sperm and mix everything gently.
10.) Add 1 mL of PLI and mix well.
11.) Transfer the DNA samples into 1.5 mL eppendorf tubes and 200 μL of the yeast culture.
12.) Heat shock the samples for 30 mins at 42 °C in a heat block.
13.) After transformation, spin down the cells at 7000RCF for 1 min at room temperature and discard the supernatant.
14.) Resuspend the pellet in 1 mL of water.
15.) Spin down the cells at 8000RCF for 1 min at room temperature.
16.) Remove 900 μL of the water and resuspend the cells in the remaining water.
17.) Finally, add the transformed cells onto the necessary plates and spread it uniformly.
18.) Incubate the plates for 3 nights at 30°C.

Western blot
![](https://static.igem.org/mediawiki/2019/f/fc/T--UCopenhagen--Arrowcollap.png)

**Reagents required:**
• Sample cultures
• Milli-Q water
• Lysis Buffer: 50 mM phosphate buffer with 1 mM PMSF
• Glass beads
• 5 M Guanidinium chloride or 8 M urea
• SDS gel
• 5x Protein loading buffer
• 1x MOPS buffer
• Precut PVDF membrane plus filter papers
• PBS-T buffer and milk powder dissolved in PBS-T buffer
• Primary antibody
• Secondary antibody
• Pierce ECL Western Blotting substrate
• Biorat prestained marker

**Procedure:**
**Protein preparation**
1.) Spin down sample culture, remove supernatant, wash pellet in Milli-Q.
2.) Spin down, resuspend pellet in 500 μL lysis buffer and transfer to Eppendorf tube containing 500 μL glass beads.
3.) Vortex tube for 1 minute, then let it chill on ice for one minute. Repeat 9 times.
4.) The layer above the glass beads is collected an centrifuged for 15 minutes at maximum speed and 4° C. The supernatant is collected: it contains soluble proteins.
5.) The pellet is resuspended in 500 μL Guanidinium chloride and boiled at 99° C for 5 minutes. After centrifugation, the insoluble protein is collected in the supernatant.
**SDS-PAGE**
1.) The SDS gel is loaded into the electrophoresis cassette and spilled with 1x MOPS buffer

2.) 40 μL of protein solution are mixed with 10 μL 5x Protein loading buffer. The samples is loaded on the SDS gel. As ladder Biorad prestained marker is used.
3.) The gel is run for 1h at 200V.
**Western Blot**
1.) The filter paper and the membrane are assembled around the gel according to the manual of the precut membrane and the filter.

2.) The stack is place in the Bio-Rad Trans-Blot Turbo machine, which is then started.
3.) After the plotting, the membrane is transferred in PBS-T milk, shaken in it for 1-2 hours at room temperature.
4.) The primary antibody is directly added into the milk in volume that results in a 5000x dilution of the antibody. The membrane is shaken for 1-2 hours at room temperature or at 4° C over night.
5.) The milk is removed and saved. Then, the membrane is washed three times for 15 minutes with PBS-T.
6.) The secondary antibody is put into PBS-T milk so a 2000x dilution of it is obtained.
7.) The membrane is incubated in milk, containing the secondary antibody and then washed as described under 5.).
8.) The membrane is developed using the Pierce ECL Western Blotting Substrate.

# About Us

We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

![](https://static.igem.org/mediawiki/2019/6/61/T--UCopenhagen--LgogForBlack.png)

# Keep in Touch

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iGEM\_Copenhagen

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iGEM\_Copenhagen

![](https://static.igem.org/mediawiki/2019/a/a2/T--UCopenhagen--Emaillogo.png)
UCPH.IGEM2019@gmail.com

# Address

University of Copenhagen

Thorvaldsensvej 40, Frederiksberg C

Denmark

![](https://static.igem.org/mediawiki/2019/7/72/T--UCopenhagen--KUlogo.png)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UCopenhagen/Results

* [![](https://static.igem.org/mediawiki/2019/0/0b/T--UCopenhagen--Onlytextlogo.png)](https://2019.igem.org/Team%3AUCopenhagen)
* Project
  + [Description](https://2019.igem.org/Team%3AUCopenhagen/Description)
  + [Design](https://2019.igem.org/Team%3AUCopenhagen/Design)
  + [Results](https://2019.igem.org/Team%3AUCopenhagen/Results)
  + [Future Plans](https://2019.igem.org/Team%3AUCopenhagen/FuturePlans)
* Lab
  + [Parts Overview](https://2019.igem.org/Team%3AUCopenhagen/Parts)
  + [Protocols](https://2019.igem.org/Team%3AUCopenhagen/Experiments)
  + [Safety](https://2019.igem.org/Team%3AUCopenhagen/Safety)
  + [Notebook](https://2019.igem.org/Team%3AUCopenhagen/Notebook)
  + [Modeling](https://2019.igem.org/Team%3AUCopenhagen/Model)
* Human Practices
  + [Integrated Human Practices](https://2019.igem.org/Team%3AUCopenhagen/Human_Practices)
  + [Entrepreneurship](https://2019.igem.org/Team%3AUCopenhagen/Entrepreneurship)
  + [App](https://2019.igem.org/Team%3AUCopenhagen/App_Description)
  + [Education and Engagement](https://2019.igem.org/Team%3AUCopenhagen/Public_Engagement)
* Collaboration
  + [Collaborations](https://2019.igem.org/Team%3AUCopenhagen/Collaborations)
  + [Meetups](https://2019.igem.org/Team%3AUCopenhagen/Meetups)
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* Team
  + [Team Members](https://2019.igem.org/Team%3AUCopenhagen/Team)
  + [Team Building](https://2019.igem.org/Team%3AUCopenhagen/Team_Building)
* Acknowledgements
  + [Attributions](https://2019.igem.org/Team%3AUCopenhagen/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3AUCopenhagen/Sponsors)
* ![](https://static.igem.org/mediawiki/2019/a/a6/T--UCopenhagen--Logo_Awards.png)

  + [Judging form](https://igem.org/2019_Judging_Form?team=UCopenhagen)
  + [Medal Requirements](https://2019.igem.org/Team%3AUCopenhagen/Medals)
  + [Awards](https://2019.igem.org/Team%3AUCopenhagen/Awards)

![](https://static.igem.org/mediawiki/2019/5/59/T--UCopenhagen--Resultsbackground.jpg)
![](https://static.igem.org/mediawiki/2019/7/7c/T--UCopenhagen--Almostblank.png)

A comprehensive summary of all the most important results and achievements by the UCopenhagen iGEM team 2019 is listed here.

Biosensor Results

[Skip to kill switch results](#Kill)

**General workflow**

![](https://static.igem.org/mediawiki/2019/a/a9/T--UCopenhagen--Labsteps.png?fbclid=IwAR1w-mVPoCnowEsFhLhgI0VLOcn-3zuradbpp206x7YGLL_tVTZdiFadRRc)

**Figure1:** Throughout the project we used the same general workflow to create our yeast strains: PCR amplification → USER ligation → *E. coli* cloning → *E. coli* colony PCR → Sequencing → Plasmid purification → Yeast transformation → Yeast colony PCR

**Design**

Our ambition was to create a chewing gum, containing a yeast biosensor, which can detect sex hormones in saliva and elicit a quantifiable response based on the levels of these. In our wet lab work, we decided to focus on creating two different biosensors with receptors for estrogen and Luteinizing Hormone (LH) respectively.
The two receptors we are using are the G-Protein-Coupled Estrogen Receptor (GPER), and the *Xenopus Laevis* Lutropin-Choriogonadotropic Hormone Receptor (XLHCGR), to detect estrogen and LH respectively. You can read more about the receptors and our general design on our [Project Design page](https://2019.igem.org/Team%3AUCopenhagen/Design).
A minimal biosensor requires five modules **(Fig. 2A)**. To construct our minimal biosensors, we chose to take advantage of the yeast pheromone response pathway. To do this, we engineered our yeast strains to include one of our receptors (GPER or XLHCGR respectively), a chimeric adaptor module, connecting the receptor to the yeast signaling pathway, and a reporter module eliciting a fluorescent response. For our signal processing module, we chose to converse the endogenous MAP kinase signaling cascade normally used in the pheromone response pathway **(Fig. 2B)**.
![](https://static.igem.org/mediawiki/2019/2/2b/T--UCopenhagen--Results1.jpg)

**Figure 2: The conserved and engineered modules of our minimal biosensor | 2A:** The required modules of a minimal biosensor. **2B:** The MAP Kinase signaling pathway already present in yeast was conserved, while the sensing module was engineered to fit a hormone receptor. The adaptor module was engineered to connect the receptor to the signaling pathway, and the reporter module was engineered to a fluorescent signal response.

**Multiplex cassette systems**

To integrate our biosensor system into the yeast genome, we used a multiplex 5-assembler genomic integration cassette (5-modular system), which allowed for integration into the yeast chromosome 10, site 3 (**Fig. 3A**). In order to confirm successful expression and localization of our receptors, we also utilized a multiplex 3-assembler genomic integration cassette (3- modular system) (**Fig. 3B**) to construct yeast strains containing our receptors linked to superfolder GFP (sfGFP). See **Figure 6** for a complete explanation of yeast strains created.
![](https://static.igem.org/mediawiki/2019/d/dc/T--UCopenhagen--modularsystems.jpg)

**Figure 3: The multiplex genomic integration system | 3A:** The 5-assembler genomic integration cassette. The first module contains the receptor. Second module contains the GPA1. Third assembler contains the transcription
factor STE12. Fourth assembler contains an empty vector backbone and the fifth assembler contains the reporter
gene ZsGreen. **3B:** The 3-assembler genomic integration cassette. The first module contains the receptor tagged
with sfGFP. The second and the third assemblers contain empty backbone vectors.

**Vector construction**

For construction of all our yeast strains we used the same general workflow. Initially all fragments were PCR amplified using primers carrying USER overhangs. The fragments were verified through gel electrophoresis (**Fig. 4A**). Following PCR amplification, the fragments, were purified and ligated into our integration vectors using USER cloning. For a detailed explanation of the backbones used, please refer to our [Project Design page](https://2019.igem.org/Team%3AUCopenhagen/Design). See **Table 1** for list of constructs.

**Table 1: The constructs made by USER ligation**

|  |  |  |
| --- | --- | --- |
| Backbone | Promoter | Gene fragment |
| X3A | pCCW12 | GPER |
| X3A | pCCW12 | XLHCGR |
| X3A | pCCW12 | GPER-linker + sfGFP |
| X3A | pCCW12 | XLHCGR-linker + sfGFP |
| Ass2A | pPGK1 | GPA1-Gαi |
| Ass2A | pPGK1 | GPA1-Gαs |
| Ass2B | pRET2 | STE12 |
| X3C | pFIG1 | ZsGreen |

Following USER cloning, successful transformant were picked and colony PCR was performed. The colony PCR products were separated by Gel electrophoresis and positive colonies were determined by comparing the band sizes on the gel with expected sizes. **Figure 4B** depicts a representative colony PCR gel electrophoresis, as performed on GPER-Li-sfGFP. Based on the results of colony PCR, 1-2 colonies were picked and grown overnight. Plasmid DNA purification was carried out and sequencing used to verify successful vector construction.
![](https://static.igem.org/mediawiki/2019/7/76/T--UCopenhagen--Figure3Results.jpg)

**Figure 4: Representative gel electrophoresis images | 4A:** PCR of USER fragments. Expected band sizes are listed for each fragment. **4B:** Colony PCR of ligated fragment pCCW12-GPER. A positive band was observed in lane 2 around 1800 bp, which correlates well to the expected band size for a construct of 1818 bp. Lane 1 does not show a band, which means that this sample was negative.

**Yeast transformation and colony PCR**

The purified vectors were transformed into S. cerevisiae, creating a total of 5 different strains (**Fig. 5**). Three strains were made using the 3-assembler cassette (OV1, OV2, OV3), and two using the 5-assembler cassette (OV4, OV5).
![](https://static.igem.org/mediawiki/2019/c/cf/T--UCopenhagen--Fig4Results.png)

**Figure 5: Overview of the 5 different cassettes transformed into yeast** | OV1 is a negative control strain transformed with empty vector backbones. OV2 and OV3 were transformed with our 2 receptors conjugated to sfGFP in module 1, and two empty vectors in module 2 and 3. OV4 and OV5 were transformed with our receptors in module 1, the adaptor GPA1 in module 2, and the transcription factor STE12 in module 3. Module 4 was kept empty in these strains, while module 5 contains the reporter ZsGreen.

Correct integration was verified using PCR. In the presence of our construct, we expect to see a band at 1000 bp, and in the absence of the constructs, we expect to see the bands at 1500 bp. For an in depth explanation of detecting positive yeast colonies, please refer to our [Project Design page](https://2019.igem.org/Team%3AUCopenhagen/Design).

**Figure 6** depicts gel electrophoresis from yeast colony PCR of the 2 strains (OV2 and OV3) transformed with our sfGFP tagged receptors. In both gel images, bands of 1000 bp were observed, which confirmed that both the receptors tagged with sfGFP have been successfully integrated into the yeast genome.
![](https://static.igem.org/mediawiki/2019/3/3a/T--UCopenhagen--2results.jpg)

**Figure 6: OV2 and OV3 yeast colony PCR gel images** | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 represent individual colonies. **6A:** Bands of the colony PCR product of yeast expressing GPER-Li-sfGFP (OV2). **6B:** Bands of the colony PCR product of yeast expressing XLHCGR-sfGFP (OV3). In both A and B, we see bands around 1 kb, confirming positively transformed yeast.

**Figure 7** depicts gel electrophoresis from yeast colony PCR of the 2 strains (OV4 and OV5) transformed with our full minimal biosensor systems. In both gel images, bands of 1000 bp were observed, which confirmed that both minimal biosensor systems containing receptors for estrogen and LH respectively, have been successfully integrated into the yeast genome.

![](https://static.igem.org/mediawiki/2019/0/03/T--UCopenhagen--fig6new.jpg)

**Figure 7: OV4 and OV5 yeast colony PCR gel images** | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 represent individual colonies. **7A:** Bands of the colony PCR product of yeast expressing the biosensor system containing GPER (OV4). **7B:** Bands of the colony PCR product of yeast expressing the biosensor system containing XLHCGR (OV5). In both A and B, we see bands around 1 kb, confirming positively transformed yeast.

**Confirmation of receptor expression and localization**
To confirm the correct expression and localization of the GPER and XLHCGR in our biosensor yeast strains (OV4 and OV5), we utilized the strains transformed with sfGFP tagged receptors (OV2 and OV3). Using these strains, we carried out western blotting with antibodies against GFP. The resulting gel images confirmed that our yeast strains were expressing the receptors (**Fig. 8, Fig 9**). Strain OV1 transformed with empty vector backbones was used as a negative control, and as expected showed no expression of sfGFP. A yeast strain constitutively expressing sfGFP was used as a positive control.
![](https://static.igem.org/mediawiki/2019/a/af/T--UCopenhagen--Resultsfig7.png)

**Figure 8: Western blot of yeast strain OV2 (GPER-Li-sfGFP) with anti-GFP | 8A:** OV2, soluble fraction. Clear bands are seen for both tested samples, as well as for the positive control. **8B:** OV2, insoluble fraction. Faint bands are seen for both tested samples, as well as for the positive control. Strain OV1 was used as a negative control and showed no expression of sfGFP.

Since the sfGFP is conjugated to the C-terminus of the receptor, GFP expression confirms the receptor expression. The protein size of the receptor is 44 kDa while the protein size of sfGFP is 27 kDa. Thus, we would expect to see a band of 71 kDa. However, we observed bands around 32 kDa. This might indicate that the receptor is expressed in a truncated form. We observed that most of the receptor was present in the insoluble fraction. This result indicates that either the receptor is located on the plasma membrane, as the insoluble parts include all membrane-bound proteins, or that the receptor, folds poorly, thus forming inclusion bodies.
![](https://static.igem.org/mediawiki/2019/4/40/T--UCopenhagen--western.png)

**Figure 9: Western blot of yeast strain OV3 (XLHCGR-sfGFP) with anti-GFP | 9A:** OV3, soluble fraction. Clear bands are seen for both tested samples, as well as for the positive control. **9B:** OV3 insoluble fraction. Faint bands are seen for both tested samples, as well as for the positive control. Strain OV1 was used as a negative control and showed no expression of sfGFP.

Expected band sizes are of 107 kDa.
XLHCGR-Li-sfGFP was found in the soluble fraction, indicating its presence within the cytosol and not anchored to the plasma membrane as expected. Also, the band size around 32 kDa indicates that the receptor might have been expressed in a truncated form.
To further examine receptor expression, confocal microscopy was performed on strains OV2 and OV3 (expressing GPER-Li-sfGFP and XLHCGR-sfGFP, respectively). Results were compared to the negative control strain OV1 (**Fig. 10** and **Fig. 11**).

![](https://static.igem.org/mediawiki/2019/7/77/T--UCopenhagen--1.png)
![](https://static.igem.org/mediawiki/2019/7/7e/T--UCopenhagen--3.jpg)
![](https://static.igem.org/mediawiki/2019/7/7e/T--UCopenhagen--2.jpg)

**Figure 10: Confocal microscopy of strains OV1 and OV2** | **10A** and **10B**: Strain OV2 expressing GPER-sfGFP, fluorescence and bright field, respectively. **10C** and **10D**: Positive control strain, constitutively expressing sfGFP, fluorescence and bright field, respectively **10E** and **10F**: Strain OV1 expressing empty vectors, fluorescence and bright field, respectively.

The confocal images above further confirmed the expression of the GPER conjugated to sfGFP in strain OV2. Furthermore, they confirmed the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptors, which is expressed inside the cell. When comparing the images of the OV2 strain to the positive control, it is evident that the receptor is localized to the plasma membrane, as we see a concentric ring in the yeast cells. As expected, no fluorescence was observed in the negative control strain OV1.

![](https://static.igem.org/mediawiki/2019/b/bb/T--UCopenhagen--4.jpg)
![](https://static.igem.org/mediawiki/2019/5/5e/T--UCopenhagen--5.jpg)
![](https://static.igem.org/mediawiki/2019/3/30/T--UCopenhagen--6.jpg)

**Figure 11: Confocal microscopy of strains OV2 and OV3 | 11A** and **11B**: Strain OV3 expressing XLHCGR-sfGFP, fluorescence and bright field, respectively. **11C** and **11D**: Positive control strain, constitutively expressing sfGFP, fluorescence and bright field, respectively **11E** and **11F**: Strain OV1 expressing empty vectors, fluorescence and bright field, respectively.

The confocal images further confirmed the expression of the XLHCGR conjugated to sfGFP in strain OV3. Furthermore, they confirmed the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptors, which is expressed inside the cell. However, from the images, intracellular localization of the XLHCGR could not be confirmed. As expected, no fluorescence was observed in the negative control strain OV1.

**Bioactivity assay: GPER biosensor**

In order to test the activity of our GPER biosensor, OV4, we carried out a bioactivity assay with strain OV4. Here, OV4 cells were induced with increasing concentrations of estradiol (estrogen), where after the fluorescent intensity was measured and compared to that of our control strain, OV1 (**Fig. 12**).
![](https://static.igem.org/mediawiki/2019/4/43/T--UCopenhagen--Fig11Results.png)

**Figure 12: Fluorescent bioactivity assay of OV4 strain** | The blue line indicates the fluorescence (RFU) from the negative control strain OV1 induced with increasing concentrations of estradiol. The orange line indicates the fluorescence (RFU) from the OV4 strain containing the minimal biosensor with GPER induced with increasing concentrations of estradiol. The Y-axis indicates RFU, while the X-axis indicates estradiol concentrations in picomolar.

As seen in **Figure 12**, no significant difference in fluorescence could be detected between the biosensor (OV4) and the negative control (OV1) when induced with estradiol. This indicated that the biosensor did not work as intended.

**Bioactivity assay: Nuclear estrogen receptor**

To further investigate the potential of a functional yeast estrogen biosensor, we ordered the Plasmid #69100 (pRS416-yZ3EV-Z3pr-yEGFP (RB3579)) from the Addgene catalog. This vector carries the transcription factor Z3EV which is activated by estrogen and induces yEGFP expression from the same plasmid. For more information on the receptor, please refer to our [Project Design page](https://2019.igem.org/Team%3AUCopenhagen/Design).
Estrogen in the nucleus should lead to an increased fluorescence compared to a control. Using the same general workflow as described above, we transformed the receptor into yeast, creating strain OV(NR). To test the functionality of the yeast strain, we set up a bioactivity assay identical to the one that was performed on strain OV4.
![](https://static.igem.org/mediawiki/2019/8/81/T--UCopenhagen--nuclear2.png)

**Figure 13: Fluorescent bioactivity assay of strain OV(NR)**| The blue lines indicates the fluorescence (RFU) from yeast transformed with empty vector, induced with increasing concentrations of estradiol. The orange line indicates the fluorescence (RFU) from the OV(NR) strain containing the Addgene Plasmid #69100, when induced with increasing concentrations of estradiol. The Y axis indicates RFU, while the X axis indicates estradiol concentrations in picomolar.

As seen in **Figure 13**, no significant difference in fluorescence could be detected between the biosensor (OV(NR)) and the negative control strain, containing an empty vector, when induced with estradiol. This indicated that the biosensor with the nuclear estrogen receptor did not work as intended, either. A possible source of error is insufficient estrogen concentration in the nucleus, or crystal formation of estrogen, as described below.
**Estrogen crystal formation**
To get a better understanding, we chose to examine our induced strain OV4 under the fluorescent microscope (**Fig. 14**). Here, we found that the estradiol formed crystals which elicit a fluorescent response. As such, these crystals might have masked any significant fluorescent responses of the cells. An option to further test the biosensors would be to perform flow cytometry, which would measure the fluorescence of each individual cell or crystal fragment. Through this method we should be able to distinguish cells from crystals, and thus filter out the crystals from the measurements. As such, we would be able to compare the fluorescent signal of cells induced with different concentrations of estradiol. However, we were not able to carry out these experiments within the timeframe of the iGEM competition.
Unfortunately, we were unable to get a hold of LH, the necessary agonist to test the functionality of the XLHCGR biosensor, expressed in strain OV5. Thus, these experiments will have to be carried out at a different time.
![](https://static.igem.org/mediawiki/2019/3/33/T--UCopenhagen--Fig13results.jpg)

**Figure 14: Fluorescence microscope image of estrogen crystal formation | 14A** and **14B:** Strain OV4, fluorescence and bright field, respectively. Here we see the estradiol forming crystals.

**Conclusions**

We successfully constructed five yeast strains using a multiplex genomic integration system. Through multiple methods, we demonstrated that the receptors are expressed, and that at least one of them is likely to locate to the plasma membrane. We can also conclude that there is a chance the receptors might be truncated, or is processed in another way, and that they might form inclusion bodies. We set up an assay to test the functionality of our biosensors, and we can conclude that the estradiol forms crystals, which interfere with the assay.

Kill Switch Results

To meet the individual consumer’s and the general public’s concern, it is important that the genetically modified yeast we are using is not able to survive outside its designated environment. To account for this, we decided that our yeast should contain a kill switch. Here, we chose to utilize the BAX-BI-1 system, which is a toxin-antitoxin system, as an initial proof of concept. Here, BAX works as a toxin inducing cell death, while BAX inhibitor-1 (BI-1) serves as an anti-toxin inhibiting the effect of BAX (Figure 15).

## Function of BAX

The way BAX induces cell death has been extensively studied and is well summarized by Westphal et al. (2011)[1](#Ref): To put it briefly, BAX naturally takes part in the intrinsic apoptosis pathway in higher eukaryotes. When the cell experiences stress, BAX is activated to oligomerize in the outer mitochondrial membrane to form pores. These enable cytochrome c to exit into the cytoplasm where it will activate caspases, important effectors of a controlled intrinsic cell death.
The yeast genome doesn’t contain any homologues of the mammalian BAX protein; this raises the question of whether yeast responds to BAX in a similar way as to what mammalian cells do. Previous studies have shown that BAX expression in yeast indeed leads to cell death even though the pathway for its activation is not present. Furthermore, yeast has been proven to be a suitable vehicle for analysis of Bcl-2 proteins, the protein family that BAX is part of[3](#Ref).

## Function of BI-1

The BAX inhibitor BI-1 can generally be regarded as promotor of cell survival. It locates to the membrane of the ER and inhibits the activation of BAX and the following pro-apoptotic effects. Besides this, BI-1 prevents cell death through BAX independent mechanisms and takes a critical role in the Ca2+ homeostasis[2](#Ref).

# Kill Switch Design

We constructed a kill switch by placing a toxin under a constitutively active promoter and the antitoxin under an inducible promoter, where the inducer is only present in the media. The yeast strain used should contain BI-1 under the control of an inducible promoter and BAX under the control of a constitutive promoter. The inducer should then be present in the designated environment; in our case the gum. As such, if the yeast escapes the gum, and therefore becomes separated from the inducer, apoptosis is induced and the yeast dies. For more information on the kill switch device, please refer to our [Project Design page](https://2019.igem.org/Team%3AUCopenhagen/Design). With this in mind, we took inspiration from the work of the 2017 iGEM team, NAU\_China 2017, and decided to set up two sets of experiments.

![](https://static.igem.org/mediawiki/2019/b/bb/T--UCopenhagen--Killlast.jpg)

**Figure 15: Overview of the BAX/BI-1 Kill switch device |** The figure depicts the genes encoding for the toxin BAX under the expression of a constitutive promoter while the anti-toxin BI-1 under the expression of an inducible promoter pGAL1. In the absence of galactose, BI-1 will not be expressed, and the toxin will be allowed to kill the yeast cell.

## Characterization

Firstly, we decided to characterize the biobrick submitted by NAU\_China 2017 (BBa\_K2365048, a BAX mutant), by expressing it in yeast under the control of the galactose inducible promoter, pGAL1. Here, we wanted to evaluate the efficiency of BAX as a yeast toxin by investigating how different concentrations of galactose in the growth media can induce cell death.

## Improvement

Secondly, we decided to improve the BI-1 biobrick (BBa\_K2365518), also submitted by NAU\_China 2017, by also placing it under the control of the GAL1 promoter. By placing BI-1 under the control of the pGAL1 transcription should only occur in the presence of galactose. We saw this improvement as necessary in order to contain the yeast in a defined environment, as a yeast strain that expresses BI-1 under control of the GAL1 promoter and BAX constitutively, will only be able to survive if enough galactose is present. If a yeast cell escapes its defined environment, the supply of this sugar will likely drop, BI-1 expression is interrupted, which will shift the toxin-antitoxin equilibrium in favour of BAX.
As we imagined the modules for the biosensor and the kill switch to be incorporated into the same yeast strain, we chose a different site for the genomic integration for the kill switch sequences, namely chromosome XI site 2.

# Vector construction

As with our biosensor, all fragments were PCR amplified with primers containing USER overhangs and sizes confirmed using gel electrophoresis. After purification of the fragments, we used USER cloning to create the constructs listed in table 2. All constructs were verified using sequencing.

**Table 2: Constructs made by USER ligation.**

| Backbone | | Promoter | Gene Fragment |
| --- | --- | --- | --- |
| 1 | XI2A | pGAL1 | BAX |
| 2 | XI2A | pADH2 | BAX |
| 3 | XI2A | pTEF1 | BAX |
| 4 | B(Ass2) | pGAL1 | BI-1 |
| 5 | XI2C | - | - |
| 6 | pUUS | pTEF1 | BAX |
| 7 | pUUS | pADH2 | BAX |
| 8 | pUUS | pGAL1 | BAX |
| 9 | pUUS | - | - |
| 10 | pWUS | pGAL1 | BI-1 |
| 11 | pWUS | pTEF1 | BI-1 |
| 12 | pWUS | - | - |

# Yeast transformation and colony PCR

As part of this study, a total of four strains (OV1, OV6 to OV8; Figure 16) were created using the 3-assembler system listed above (Figure 3), with insertion into chromosome 11. For all transformations yeast colony PCR was used to confirm correct chromosomal integration (Figure 17).

![](https://static.igem.org/mediawiki/2019/5/52/T--UCopenhagen--KS2.png)

**Figure 16: Overview of the 3 different cassettes transformed into yeast.**

![](https://static.igem.org/mediawiki/2019/7/75/T--UCopenhagen--KS3.jpg)

**Figure 17: Yeast colony PCR of the transformed constructs.** Specific yeast genotyping primers were used for the PCR reaction. We expected bands around 900 bp for positive colonies and 1500 bp for untransformed colonies. Unfortunately, there were no positive colonies for the pADH2-BAX+pGAL1-BI-1 construct (not shown).

# Galactose induction assay

To analyse the effect of BAX on our yeast under different expression levels, we conducted a galactose induction assay using raf-U plates with five different galactose concentrations.
Cultures of yeast containing pGAL1-BAX (OV6) or empty vector (OV1) were grown O/N and then diluted to an OD600nm of 0.5. On plates with either 0%, 0.025%, 0.05%, 0.1% or 0.2% galactose, 10 µl of each culture were spotted in increasing dilutions (10-1 to 10-4; Figure 18 and 19). After three days of incubation at 30 °C, two observations were made. First of, the strain with the integrated pGAL1-BAX construct showed decreased growth compared to the control strain even when galactose was absent (Figure 18). This suggests that the galactose promoter is leaky and a low amount of BAX is produced at all times.

![](https://static.igem.org/mediawiki/2019/c/c0/T--UCopenhagen--KS4.png)

**Figure 18: Growth of OV1 and OV6 in the absence of galactose.** The cultures were spotted in the dilutions 10-1 to 10-4 of an OD600nm of 0.5 and incubated for three days at 30 °C.

Secondly, when comparing the spots of OV6 at a dilution of 10-1 at different galactose concentrations, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed. This suggests that successful induction of BAX leads to apoptosis in our yeast.

![](https://static.igem.org/mediawiki/2019/8/8d/T--UCopenhagen--KS5.png)

**Figure 19: Growth comparison of OV6 and control strain OV1 in the presence of varying galactose concentrations.** Shown are the CFUs at an OD of 0.05 after incubation at 30 °C for four days. The three yellow colonies seen at 0.2% galactose in OV6 can be morphologically distinguished from the others, suggesting that they are contaminants.

# Quantitative galactose induction assay of pGAL1-BAX

To further analyze the effect of BAX on our yeast under different expression levels, we conducted a quantitative galactose induction assay. Here, an O/N culture of yeast containing pGAL1-BAX (OV6) was diluted to an OD600nm of 0.5. Subsequently, 100 µl of the culture in dilutions of 10-3 and 10-4 were spread on plates with 0%, 0.05%, 0.1%, 0.3% and 1% galactose, respectively. Each plate was made in duplicates. After incubation for three days at 30 °C, the CFU/ml were calculated and compared to the control (Figure 20).

![](https://static.igem.org/mediawiki/2019/a/a9/T--UCopenhagen--KS6.png)

**Figure 20: Gradient induction of BAX in yeast using the inducible GAL1 promoter.** Shown are the CFUs per ml that have resulted from cultures with an OD600nm of 0.5 in presence of varying galactose concentrations (0%, 0.05%, 0.1%, 0.3% and 1%). If no errorbar is indicated, only one of the used duplets showed a quantifiable amount of colonies.

As seen on figure 20, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately, our control strain (OV1) was only plated on raf-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to OV6 grown in 0 % galactose media).
In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this indicates that the cells were killed before a normal sized colony could be formed. To test this, the OV6 plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed (Figure 21).

![](https://static.igem.org/mediawiki/2019/1/12/T--UCopenhagen--KS7.png)

**Figure 21: Comparison of colony sizes of OV1 and OV6 grown on raf-U agar with galactose.** Left: Colonies of the control strain OV1 (dilution 10-3 ) in the presence of 1% galactose after incubation for three days at 30 °C. Right: Colonies of the pGAL1-BAX strain OV6 (dilution 10-3) in the presence of 0.1% galactose after incubation for four days at 30 °C.

# Dual Plasmid transformation

In addition to the strains above, an additional set of strains were constructed using yeast expression plasmids (Figure 22).

![](https://static.igem.org/mediawiki/2019/9/91/T--UCopenhagen--KS.png)

**Figure 22: Schematic overview of the plasmids the strains OV9-18 were transformed with.**

Following transformation, the yeast cultures were plated. However, the transformed strains only grew partly as expected and later we encountered problems cultivating them in liquid media, which is why we decided against carrying out further experiments with them.

# BAX rescue assay

We wanted to examine whether BI-1 is able to neutralize the lethal impact of BAX, and therefor performed a rescue assay. In this, both BAX and BI-1 were put under control of the GAL1 promoter (OV19).
For construction of the strain, we transformed the OV6 strain (pGAL1-BAX integrated into the genome) with a high-copy number pGAL1-BI-1 plasmid.
For the assay colonies were picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalised to 0.035 and different dilutions of the colonies (10-0 to 10-3) were spotted on plates with Glu-UW agar and raf-U-W agar with 1% galactose in volumes of 10 µl. A yeast strain containing only pGAL1-BAX and the empty pWUS plasmid served as control (OV20) and was spotted along with the sample (Figure 23).

![](https://static.igem.org/mediawiki/2019/7/70/T--UCopenhagen--Bax_rescue.png)

**Figure 23: BAX rescue assay.**
In this assay, the strains OV19 and OV20 were grown on both glu-U-W agar (left) and raf-U-W (1% galactose) agar in different dilutions of OD600nm (10-0 to 10-3).

As BAX was expressed under the inducible promoter pGAL1 in both strains, we expected normal growth on the glu-U-W plates. On the raf-U-W plates with 1% galactose, we expected the control strain OV20 to show a decreased colony size and number compared the strain OV19 that should be rescued by the BI-1 plasmid. However, as seen in figure 23, no significant growth was observed for either strain in the presence of galactose. As such, we saw no indication that BI-1 was able to prevent BAX induced apoptosis. While it is possible that the lack of function of BI-1 could be the result of a loss of function during transformation, our data suggests that BI-1 is not able to prevent BAX induced apoptosis in our system, and therefore is not suitable for our kill switch.

# References

1. Westphal, D., Dewson, G., Czabotar, P. E., Kluck, R. M. (2011): Molecular biology of BAX and Bak activation and action. Biochimica et biophysica acta 1813 (4), pp. 521–531.

2. Li, B., Yadav, R. K., Jeong, G. S., Kim, H-R., Chae, H-J. (2014): The characteristics of BAX inhibitor-1 and its related diseases. Current molecular medicine 14 (5), pp. 603–615.

3. Polčic, P., Jaká, P., Mentel, M. (2015): Yeast as a tool for studying proteins of the Bcl-2 family. Microbial cell (Graz, Austria) 2 (3), pp. 74–87.

# About Us

We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

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