iGEM 2017 — Team UiOslo_Norway

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# Team:UiOslo Norway/Notebook

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# Notebook

#### [February](#collapseOne)

Team building activities

#### [March](#collapseTwo)

Team Building and Discussion on project ideas
Attended the BioBrick Tutorial 2017 with several other Scandinavian iGEM-teams at the DTU.
**Hilde**: Joined the team.
**Elisabeth**: Joined the team.
**Aman**: Joined the team. Atten Biobrick tutorial at DTU.

#### [April](#collapseThree)

Discussion with SMEs from Physics and Molecular Biology for background information on feasibility of Bilogical Lasers
**Can**: Joined the team.
**Dejana**: Joined the team.
**Kristina**: Joined the team.

#### [May](#collapseFour)

Laboratory work: expressing and purifying his-tagged GFP.
**Dejana**: Participated in purifying sfGFP isolated from *E. coli*.
**Can**: Participated in purifying sfGFP isolated from *E. coli*.
**Eirik**: Participated in laboratory work on the 15th and the 22nd of May.
**Hilde**: Did some research/talking to professors regarding what kind of equipment we needed for the biolaser.
**Helene**: Joined the team.
**Kristina**:Participated in lab work regarding sfGFP isolation.
**Aman**: Worked on wiki template

#### [June](#collapseFive)

**Eirik**: worked on designing parts and primers for the project. Participated in labwork.
**Hilde**: Did some more research/talking to professors regarding what kind of equipment we needed for the biolaser.
**Elisabeth**: Research regarding equipment and started looking around for possible sponsors.
**Helene**: Started to understand what the project was all about and got updated on what had happened since February.
**Kristina**: Finished mandatory tour of the lab and safety requirements to be able to work in the lab.
**Dejana**: Finished mandatory tour of the lab and safety requirements to be able to work in the lab.
**Can**: Finished mandatory tour of the lab and safety requirements to be able to work in the lab.
**Aman**: Held presentation for professors and researchers at the Science Library.

#### [July](#collapseSix)

**Eirik**: Participated in labwork for the InterLab Measurement Study and for molecular cloning.
 **Hilde**: Made a LED as lab equipment with help from Nils-Olav Skeie, wrote some modelling, also contacted Alex regarding using his lab
 **Elisabeth**: Attended the physics lab, where we worked on our current set-up. Trying to reach out to Thorlabs at this point, to get the right equipment.
**Helene**: Started the search for equipment for the physics lab, and sent a BUNCH of e-mails to every possible laser owner in Oslo.
 **Kristina**:Worked in the lab with insertion of nmt1 and cyc1 into submission vector.
 **Aman** : Worked on wiki design.

#### [August](#collapseSeven)

**Can**: Made github for iGEM tools, and program to search the iGEM registry for parts.
**Eiri**k: Participated in labwork for cloning, protein exta and for the InterLab Measurement Study. Held presentations for new physics students and new bioscience master students.
**Hilde**: did some more phys-lab, also arranged and held the presentation for the new physics students with Eirik
**Elisabeth**: Got in touch with Thorlabs, who agreed to sponsor us. Also had a meeting with another possible sponsor, as well as acquiring sequencing for DNA from GATC. Worked at the physics lab testing GFP with the current setup. Started running the social media pages.
**Helene**: Managed to borrow color filters and a CCD-camera from the Institute of Theoretical Astrophysics. Tested a GFP sample with our current setup.
**Kristina** : Participated in labwork for cloning, protein exta and for the InterLab Measurement Study.
**Dejana** : Participated in labwork for the InterLab Measurement Study.
**Aman** : Continued work on the wiki.

#### [September](#collapseEight)

**Eirik**: Joined for the NTNU workshop. Participated in molecular cloning labwork, miniprepping, PCR, gel analysis and sequencing setup.

 **Hilde**: More phys-lab while also writing for the wiki and some simple illustrations for the wiki
 **Elisabeth**: Held a practice presentation with Elena to get feedback for the project. Worked at the physics lab while figuring out which equipment we needed from Thorlabs. Running facebook-page with more updates on the team.
**Helene**: Did more lab work and made more LED-circuits with red and green light. Tested the GFP with our modified setup and with the different LED-circuits. Presented our project at the Cutting Edge Festival at UiO.
 **Kristina**:Worked in the lab with insertion of nmt1 and composite part into submission vector. Took part in NTNU workshop.
 **Dejana** :Worked in the lab with insertion of nmt1, cyc1 and composite part into submission vector. Took part in NTNU workshop.
 **Can** :Worked in the lab with insertion of nmt1, cyc1 and composite part into submission vector. Took part in NTNU workshop.
 **Aman**: More work on wiki. Talked to Center for Digital Life Norway regarding sponsorship. Participated in workshop at NTNU.

#### [October](#collapseNine)

**Eirik**: Presented for the minister of education. Participated in molecular cloning labwork, miniprepping, PCR, gel analysis and sequencing setup. Very active in writing to the wiki
**Aman**: Organized wiki-workshop every week or twice a week. Added large amounts of wiki content, as well as editing and formatting a large amount of the content others wrote. Wrapped up the wiki work with assistance of the team.
 **Hilde**: Final phys-lab work, also worked on getting some merchandise for the group. Actively participated in the wiki writing.
**Elisabeth**: Finally received equipment from Thorlabs. Final physics lab work, now also testing yeast in addition to more GFP. Contacting sponsors in order to get logos and how they wish to be represented. More social media. Actively participated in wiki writing.
**Helene**: Finalized the setup and tested it with GFP and yeast. Did the final lab work and wrote on the wiki.
 **Kristina**:Worked in the lab with insertion of nmt1 and composite part into submission vector. Testing out modifications of protocols. Wrote parts for the wiki.
 **Dejana** :Worked in the lab with insertion of composite part into submission vector. Wrote parts for the wiki. Prepared yeast cells for the biolaser. Worked on collaboration with Hefei University of Technology.
 **Can** :Worked in the lab with insertion composite part into submission vector and shipping. Worked on the Wiki. Worked with transformation of yeast cells.
 **Aman** :

[Problemveien 7, Oslo, Norway](https://www.google.no/maps/place/Problemveien%2B7%2C%2B0313%2BOslo/%4059.9401331%2C10.7180943%2C17z/data%3D%213m1%214b1%214m5%213m4%211s0x46416de7a8e9c1a1%3A0x917a1b33171c58b1%218m2%213d59.9401331%214d10.720283)

uioslonorway@gmail.com

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# Team:UiOslo Norway/Experiments

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# 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)

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# Team:UiOslo Norway/Results

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  + [Public Engagement](https://2017.igem.org/Team%3AUiOslo_Norway/Engagement)
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  + [Collaborations](https://2017.igem.org/Team%3AUiOslo_Norway/Collaborations)
  + [Sponsors](https://2017.igem.org/Team%3AUiOslo_Norway/Sponsors)
* Team
  + [Team Members](https://2017.igem.org/Team%3AUiOslo_Norway/Team)
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  + [Achievements](https://2017.igem.org/Team%3AUiOslo_Norway/Achievements)

# Results

* [Physics](#tab-item1)
* [Parts in submission vectors](#tab-item2)
* [sfGFP purification](#tab-item3)
* [NTNU collaboration](#tab-item4)
* [InterLab](#tab-item5)

Our results vary from the characteristics of light from a LED to how a protein solution with fluorescent properties (GFP) act when light upon:

* We managed to create a light source that emitted light in the correct wavelength for the GFP to get excited. This being a homemade LED-circuit with a blue diode.
* We proved that there were no fluorescens from the GFP when light upon with green or red light, only when being light upon by the light source that had the correct wavelength (blue LED-circuit).
* We managed to verify that you do not need monochromatic light to get fluorescens.
* We were not able to measure the light emitted from the GFP with a CCD-camera.
* We got to test multiple setups with the GFP solution, but none proved lasing, at least not with our equipment.
* In the end we had some red light emitted from the GFP used, this we could see in the spectrometer.

![](https://static.igem.org/mediawiki/2017/0/07/T--UiOslo_norway--results_sGFPworking.JPG)

sfGFp sample that had fluorescence with our set up

![](https://static.igem.org/mediawiki/2017/b/bf/T--UiOslo_norway--results_broken_sfgfp.JPG)

sfGFP that emitted red light.

![](https://static.igem.org/mediawiki/2017/1/17/T--UiOslo_norway--results_sGFPredLED.JPG)

Testing the same sfGFP as above with red light, no fluorescence.

![](https://static.igem.org/mediawiki/2017/4/43/T--UiOslo_norway--results_sGFPspectrum.JPG)

The light spectrum for the LED with no filters, a 4000 spaces pr inch. Showing that the light is not monochromatic.

![](https://static.igem.org/mediawiki/2017/1/15/T--UiOslo_norway--results_sGFPbroken.JPG)

One of the sfGFP sample that did not work

![](https://static.igem.org/mediawiki/2017/2/2b/T--UiOslo_norway--results_sGFPgreenLED.JPG)

Testing the same sfGFP as aboce with green light, still no fluorescence.

### CYC1 / [BBa\_K2110000](http://parts.igem.org/Part%3ABBa_K2110000)

Successful transformation of E.coli TOP10 with Gibson solution indicated by bacterial growth on LB plates containing chloramphenicol(25 mg/mL).

Results from PCR further confirm an insert of correct length.

Sequencing results show as successful insertion into pSB1C3. [Sequencing data provided here](https://static.igem.org/mediawiki/2017/a/a7/T--UiOslo_norway--results_cyc1_sequence_7IJ95.txt). Has a 100% identity with BBa\_K2110000.

### Nmt1 / [BBa\_K2424000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2424000):

Successful transformation of E.coli TOP10 with Gibson solution indicated by bacterial growth on LB plates containing chloramphenicol(25 mg/mL). However there were red colonies persent, [meaning that they contained an empty vector.](http://parts.igem.org/Help%3AProtocols/Linearized_Plasmid_Backbones)

Gel from PCR suggested that white colonies do not contain insert, but rather are bacteria that are not chloramphenicol resistant. This occurs when plates are incubated to long and antibiotica has deteriorated. Reason for longer incubation time than recommended is due to the antibiotics used. Chloramphenicol is tougher on bacteria cells than ampicillin and bacteria may need more time to grow.

Several attempts have given same result. It been theorized that the reason why nmt1 is harder to insert is caused by its DNA secondary structure; we were informed that this could potentially cause problems when the DNA was syntesized.

However, this stepback has caused us to modify gibson assembly protocol. Increase incubation to 1h and dilute the gibson solution to remove some molecules in the Gibson Mix causing trouble during transformation. We have diluted 1:3, but increased the volume used in transformation to 3 times of original volume. Leading to the same volume of plasmid, but ⅓ of the gibson mix. Results show an increased number of transformed colonies from the diluted solution.

Further results for PCR indicate a insert of right size. Samples have also been sent to GATC for sequencing.

### Composite part :

We faced similar problems with the composite part as with NMT1; likely for the same reasons as above, as the composite part contains the NMT1 sequence and would face the same issues with the secondary structure. We ordered specific primers to create a composite part with 5A overhangs (see Lab Methods) and used these to do 3A assembly on the composite part. This eventually lead to a successful transformation, and this transformation was purified and sent for sequencing.
![](https://static.igem.org/mediawiki/2017/2/2d/T--UiOslo_norway--results_nmt1_fail.JPG)

![](https://static.igem.org/mediawiki/2017/8/88/T--UiOslo_norway--Project-Lab--Gel_Compositpart_fail.JPG)

![](https://static.igem.org/mediawiki/2017/b/bb/T--UiOslo_norway--Project-Lab--Gibson_Diluted.JPG)

![](https://static.igem.org/mediawiki/2017/a/a2/T--UiOslo_norway--Project-Lab--Cyc1.JPG)

![](https://static.igem.org/mediawiki/2017/6/65/T--UiOslo_norway--fragmnt_cyc.jpg)

![](https://static.igem.org/mediawiki/2017/e/e1/T--UiOslo_norway--Project-Lab--Gel_nmt1.JPG)

Using this [calculator](http://www.biomol.net/en/tools/proteinextinction.html), we calculated the extinction coefficient and molecular weight:

The same calculations are done for batch nr. 2 (1 mL) and batch nr. 3 (877µL), which are actually samples from column nr. 2, just split into two. The results for these samples are:

* batch nr. 2 (1 mL): 5.15 mg
* batch nr. 3 (877µL): 10.9 mg

This gives total mass of:
total mass (sfGFP)=19.3 mg+5.15 mg+10.9 mg=35.35 mg

**SDS-page**
**Observations**
Batch nr. 1 had bright green colour, and we got a lot of precipitation in this solution after dialysis.
Batch nr. 2 and batch nr. 3 are made out of one sample from dialysis. It was not resuspended after centrifugation so the batch nr. 2 and 3 have different concentrations of sfGFP and amount of precipitation (batch nr. 3 had more precipitation and bigger absorbance).

### Second experiement

**Observations**
The product was green which indicates presence of sfGFP protein.

![](https://static.igem.org/mediawiki/2017/d/d5/T--UiOslo_norway--Project-Results_fig6.jpg)

The isolated sfGFP stored in test tubes at 4ºC after repeating the procedure

![](https://static.igem.org/mediawiki/2017/c/c7/T--UiOslo_norway--Project-Results_fig3-calculations.JPG)

Calculation of the extinction coefficient and molecular weight for sfGFP

![](https://static.igem.org/mediawiki/2017/7/7f/T--UiOslo_norway--Project-Results_fig2-results.jpg)

![](https://static.igem.org/mediawiki/2017/a/af/T--UiOslo_norway--Project-Results_fig5.jpg)

The gel after 1 hour of distaining (SDS-Page ran for 2x40min)

[Fig. 2.0.](https://static.igem.org/mediawiki/2017/b/b0/T--UiOslo_norway--results_NTNU_collab.jpg) Significant differences observed between number of colonies formed (pLA230/MP6 transformed Top10 Ecoli cells) in plates, pre-incubated with either 20 mM arabinose or glocose in day3. Test (+ Amp, down) and control (-Amp, up). Individual plate results shown below. The images are linked rather than put here as the colonies are small and we need a very high resolution to see the colonies properly:
[Fig. 2.1.](https://static.igem.org/mediawiki/2017/c/c8/T--UiOslo_norway--results_collab_0.jpg) 10^0 concentration (+Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.2.](https://static.igem.org/mediawiki/2017/8/83/T--UiOslo_norway--results_collab_-1.jpg) 10^-1 concentration (+Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.3.](https://static.igem.org/mediawiki/2017/6/6a/T--UiOslo_norway--results_collab_-2.jpg) 10^-2 concentration (+Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.4.](https://static.igem.org/mediawiki/2017/8/8a/T--UiOslo_norway--results_collab_-3.jpg) 10^-3 concentration (+Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.5.](https://static.igem.org/mediawiki/2017/e/ef/T--UiOslo_norway--_results_collab_-5.jpg) 10^-5 concentration (-Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.6.](https://static.igem.org/mediawiki/2017/2/26/T--UiOslo_norway--results_collab_-6.jpg) 10^-6 concentration (-Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.7.](https://static.igem.org/mediawiki/2017/2/26/T--UiOslo_norway--results_collab_-7.jpg) 10^-7 concentration (-Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.
[Fig. 2.8.](https://static.igem.org/mediawiki/2017/2/26/T--UiOslo_norway--results_collab_-8.jpg) 10^-8 concentration (-Amp); colonies formed pre-incubated with either 20 mM arabinose or glocose in day3.

**Colony count for individual tests**
Test\_10^0: Ara.~8000 Glu.=400
Test\_10^-1: Ara.=800 Glu.=120
Test\_10^-2: Ara.=600 Glu.=10
Test\_10^-3: Ara.=181 Glu.=4

Control\_10^-5: Ara.=125 Glu.=30
Control\_10^-6: Ara.=30 Glu.=11
Control\_10^-7: Ara.=3 Glu.=4
Control\_10^-8: Ara.=0 Glu.=1

Results from the InterLab Study can be found at the [InterLab](https://2017.igem.org/Team%3AUiOslo_Norway/InterLab) page of our wiki.

[Problemveien 7, Oslo, Norway](https://www.google.no/maps/place/Problemveien%2B7%2C%2B0313%2BOslo/%4059.9401331%2C10.7180943%2C17z/data%3D%213m1%214b1%214m5%213m4%211s0x46416de7a8e9c1a1%3A0x917a1b33171c58b1%218m2%213d59.9401331%214d10.720283)

uioslonorway@gmail.com

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