iGEM 2019 — Team Stockholm

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

# Team:Stockholm/Notebook

<!doctype html>iGEM Stockholm Notebook

[![](https://static.igem.org/mediawiki/2019/7/70/T--Stockholm--esther-white.svg)](/Team%3AStockholm)

Project
[Description & Inspiration](/Team%3AStockholm/Description) [Design](/Team%3AStockholm/Design) [Notebook](/Team%3AStockholm/Notebook) [Results](/Team%3AStockholm/Results) [Model](/Team%3AStockholm/Model) [Demonstrate](/Team%3AStockholm/Demonstrate) [Outlook](/Team%3AStockholm/Outlook/) [Safety](/Team%3AStockholm/Safety) [Contribution](/Team%3AStockholm/Contribution)

Parts
[Parts](/Team%3AStockholm/Parts) [Primers](/Team%3AStockholm/Primers)

Human
Practices[Integrated Human Practices](/Team%3AStockholm/Human_Practices) [Public Engagement](/Team%3AStockholm/Public_Engagement) [Art Exhibition](/Team%3AStockholm/Art_Exhibition) [Documentary](/Team%3AStockholm/Documentary)

About
[Team](/Team%3AStockholm/Team) [Attributions](/Team%3AStockholm/Attributions) [Collaborations](/Team%3AStockholm/Collaborations) [Sponsors](/Team%3AStockholm/Sponsors) [Esther Lederberg](/Team%3AStockholm/Esther_Lederberg)

Awards
[Software](/Team%3AStockholm/Software) [Entrepeneurship](/Team%3AStockholm/Entrepeneurship) [Medal Criteria](/Team%3AStockholm/Medal_Criteria)

# Notebook

> From early June to late October we have worked hard in the lab to realize Esther. Here, you can find all of the protocols used, a timeline of important points in the process, as well as the lab notes themselves.

## Dictionary

During our laboratory work, we assigned nicknames to our BioBricks for the sake of simplicity. This table will allow the reader to understand what BioBrick we are mentioning in our notebook.

| Notebook name | Wiki name | Registry Link |
| --- | --- | --- |
| BB1 | C-protein Characterization Construct (Lysogenic module) | [http://parts.igem.org/Part:BBa\_K3024004](http://parts.igem.org/Part%3ABBa_K3024004) |
| BB1.2 | Inducible C-protein construct - Regulate lysogenic module | [http://parts.igem.org/Part:BBa\_K3024009](http://parts.igem.org/Part%3ABBa_K3024009) |
| BB2 | Inducible cox characterization construct (Lytic module) | [http://parts.igem.org/Part:BBa\_K3024011](http://parts.igem.org/Part%3ABBa_K3024011) |
| BB3 | Phage genome simulation module | [http://parts.igem.org/Part:BBa\_K3024012](http://parts.igem.org/Part%3ABBa_K3024012) |

## Protocols

### PCR

[Taq Colony PCR Protocol.pdf](https://static.igem.org/mediawiki/2019/1/10/T--Stockholm--Taq_Colony_PCR_Protocol.pdf)

[Q5 PCR Protocol.pdf](https://static.igem.org/mediawiki/2019/1/1f/T--Stockholm--Q5_PCR_Protocol.pdf)

### Digestion

[Digestion Protocol AraC3 Vector and BioBricks.pdf](https://static.igem.org/mediawiki/2019/9/94/T--Stockholm--Digestion_Protocol_AraC3_Vector_and_BioBricks.pdf)

[Digestion Protocol C3 Vector and BioBricks.pdf](https://static.igem.org/mediawiki/2019/e/eb/T--Stockholm--Digestion_Protocol_C3_Vector_and_BioBricks.pdf)

### Ligation and Transformation

[Ligation and Transformation Protocol AraC3 and C3 vectors.pdf](https://static.igem.org/mediawiki/2019/7/70/T--Stockholm--Ligation_and_Transformation_Protocol_AraC3_and_C3_vectors.pdf)

### HIFI Cloning

Manufacturers protocol:

[NEBuilder HiFi DNA Assembly Reaction Protocol](https://international.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)

Alterations on the manufacturers protocol:

Create inserts with complementary ends to the vector, by either amplifying the vector or the inserts. a Total amount of max 200fmol DNA, with 50ng of Vector, is mixed with the HIFI master mix and incubated at 50°C for 15 minutes. Of the 20µl ligation reaction, 2µl were mixed with 6µl of water and 2µl 5x KCM. After one minute on Ice, this mix was used for transformation by heat-shock with 10µl of KCM competent cells.

[HIFI cloning.pdf](https://static.igem.org/mediawiki/2019/0/04/T--Stockholm--HIFI_cloning.pdf)

### Fluorescence spectrometry

[Fluorescence Spectrometry Method.pdf](https://static.igem.org/mediawiki/2019/c/c6/T--Stockholm--Fluorescence_Spectrometry_Method.pdf)

### Flow cytometry

[Flow Cytometry Protocol.pdf](https://static.igem.org/mediawiki/2019/7/73/T--Stockholm--Flow_Cytometry_Protocol.pdf)

### Absorbance spectrometry

[Absorbance Spectrometry.pdf](https://static.igem.org/mediawiki/2019/4/47/T--Stockholm--Absorbance_Spectrometry.pdf)

### KCM Competent Cells

[KCM Competent Cells Protocol.pdf](https://static.igem.org/mediawiki/2019/7/75/T--Stockholm--KCM_Competent_Cells_Protocol.pdf)

### Western blot

[Western blot Protocol.pdf](https://static.igem.org/mediawiki/2019/0/0b/T--Stockholm--Western_Blot.pdf)

### Overlap Extension

[OE-PCR protocol.pdf](https://static.igem.org/mediawiki/2019/3/3b/T--Stockholm--OE-PCR_protocol.pdf)

[Overlap Extension Protocol.pdf](https://static.igem.org/mediawiki/2019/6/69/T--Stockholm--Overlap_Extension_Protocol.pdf)

Q5 Site Directed Mutagenesis

[Q5® Site-Directed Mutagenesis Kit Protocol (E0554)](https://international.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554)

## Workflow

### June

* 12/6 p15A with kanamycin resistance was chosen as backbone.
* 12/6 Improved BioBricks with RBS and Terminators were ordered.
* 26/6 Improved BioBricks arrived with primers that will insert overhangs past the prefix and suffix.
* 27/6 Altered growth curve protocol for E.coli C1757 to reduce chance of contamination.
* 28/6 Ordered primers that add basepairs past the prefix and suffix, to facilitate better binding sites for restriction enzymes.

### July

* 1/7 Switch from p15A to the C3 and C5 plasmids that have chloramphenicol instead of kanamycin and are easier to handle.
* 5/7 Successfully created competent C1757 cells using the KCM protocol.
* 6/7 Ligation with T4 ligase for 10 min according to the manufacturer protocol did not work.
* 8/7 Brick 1.2 and Brick 3 are amplified but not brick 1 and 2, we ordered more specific primers. An advisor suggested including AraC in constructs containing pBAd.
* 9/7 New primers for overlap extension ordered and will a different cloning method will be tried.
* 10/7 We were advised to do larger PCR reactions, not to purify after digestion, use T4 ligase at 16C overnight and not to exceed the total reaction volume of 20ul and to try different molar rations. And to use much bigger volumes of insert and vector.
* 15/7 AraC3 was isolated from The iGEM kit, to be used as the standard cloning vector.
* 16/7-22/7 We tried to clone all BioBricks into either the C3 or AraC3 vector in 1:3 ratio and 1:5 ratio and there is a lot of background signaling.
* 18/7 Charactarization started. Sequencing results revealed that we had not inserted our desired brick.
* 25/7 Used Phusion ad Pfu polymerases to do colony pcr. Pfu PCR was unsuccessful at the chosen annealing temperature. All colonies were negative.
* 31/7 Added an initial cell lysis step of colonies to the Colony PCR protocol.
* 31/7 In order to reduce the background signaling, we have tried to dephosphorylate our vector.

### August

* 5/8 Successfully cloned brick 1.2 into AraC3 thanks to dephosphorylation.
* 13/8 Adding a second BioBrick to the previously created construct failed. Expected it to be caused by non-specifically digested BioBricks.
* 19/8 Time of digestion decreased from 2h to 15 min to minimize star activity.
* 26/8 Gelelectrophoresis of BB3 suddenly produces some shorter unspecific bands.

### September

* 3/9 Some of the previous positive colonies of AraC3 BB1.2, were cultured and the plasmids isolated. After sequencing, it appeared there was an extensive deletion in the RFP sequence and 2-3 new stop codons had appeared.
* 9/9 One of our advisors recommended that we change the cloning assembly technique, as well as to refrain from doing overlap extension since it is less likely you will get it to work with short overlaps. We started working with Hi-Fi cloning.
* 14/9 To reduce the amount of background the template concentration was reduced.
* 16/9 We successfully cloned BB1.2 and the Switch Plasmid. Low background noise visible.
* 20/9 Cloning of Model Plasmid not successful due to unspecific PCR products in both inserts.
* 23/9 Sequencing confirmed successful cloning of Switch Plasmid.
* 26/9 Created new inserts for cloning of Model Plasmid.

### October

* 3/10 Site-directed mutagenesis in Switch Plasmid to create BB1 and BB22 in separate plasmids.
* 4/10 Screening of colonies of BB1 and BB2
* 8/10 Sequencing confirmed that the sequences of BB1, BB2, BB1.2, BB3, and AraC3 were correct. The sequencing of the Model Plasmid showed variations on the expected sequence.
* 10/10 Adding full RBS to BB1 and BB2.
* 16/10 Construct arrived and was transformed
* 18/10 Flow Cytometry and western blot were performed on induced (Arabinose 0.2%) and non-induced BB1, BB2, Switch Plasmid, AraC3.

## Detailed lab notes

[Notebook Plasmid A and Plasmid B Characterization (Benchling).pdf](https://static.igem.org/mediawiki/2019/8/8e/T--Stockholm--notebook-plasmid-a-and-plasmid-b-characterization.pdf)

[HiFI cloning (Benchling).pdf](https://static.igem.org/mediawiki/2019/3/31/T--Stockholm--notebook-hifi-cloning.pdf)


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

# Team:Stockholm/Experiments

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3AStockholm)
[HOME](https://2019.igem.org/Team%3AStockholm)

TEAM

[Team Members](https://2019.igem.org/Team%3AStockholm/Team)
[Collaborations](https://2019.igem.org/Team%3AStockholm/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AStockholm/Description)
[Design](https://2019.igem.org/Team%3AStockholm/Design)
[Experiments](https://2019.igem.org/Team%3AStockholm/Experiments)
[Notebook](https://2019.igem.org/Team%3AStockholm/Notebook)
[Contribution](https://2019.igem.org/Team%3AStockholm/Contribution)
[Results](https://2019.igem.org/Team%3AStockholm/Results)
[Demonstrate](https://2019.igem.org/Team%3AStockholm/Demonstrate)
[Improve](https://2019.igem.org/Team%3AStockholm/Improve)
[Attributions](https://2019.igem.org/Team%3AStockholm/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AStockholm/Parts)
[Basic Parts](https://2019.igem.org/Team%3AStockholm/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AStockholm/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AStockholm/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AStockholm/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AStockholm/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AStockholm/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AStockholm/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AStockholm/Hardware)
[Measurement](https://2019.igem.org/Team%3AStockholm/Measurement)
[Model](https://2019.igem.org/Team%3AStockholm/Model)
[Plant](https://2019.igem.org/Team%3AStockholm/Plant)
[Software](https://2019.igem.org/Team%3AStockholm/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Stockholm)

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

# Team:Stockholm/Results

<!doctype html>iGEM Stockholm Results

[![](https://static.igem.org/mediawiki/2019/7/70/T--Stockholm--esther-white.svg)](/Team%3AStockholm)

Project
[Description & Inspiration](/Team%3AStockholm/Description) [Design](/Team%3AStockholm/Design) [Notebook](/Team%3AStockholm/Notebook) [Results](/Team%3AStockholm/Results) [Model](/Team%3AStockholm/Model) [Demonstrate](/Team%3AStockholm/Demonstrate) [Outlook](/Team%3AStockholm/Outlook/) [Safety](/Team%3AStockholm/Safety) [Contribution](/Team%3AStockholm/Contribution)

Parts
[Parts](/Team%3AStockholm/Parts) [Primers](/Team%3AStockholm/Primers)

Human
Practices[Integrated Human Practices](/Team%3AStockholm/Human_Practices) [Public Engagement](/Team%3AStockholm/Public_Engagement) [Art Exhibition](/Team%3AStockholm/Art_Exhibition) [Documentary](/Team%3AStockholm/Documentary)

About
[Team](/Team%3AStockholm/Team) [Attributions](/Team%3AStockholm/Attributions) [Collaborations](/Team%3AStockholm/Collaborations) [Sponsors](/Team%3AStockholm/Sponsors) [Esther Lederberg](/Team%3AStockholm/Esther_Lederberg)

Awards
[Software](/Team%3AStockholm/Software) [Entrepeneurship](/Team%3AStockholm/Entrepeneurship) [Medal Criteria](/Team%3AStockholm/Medal_Criteria)

# Results

To translate our idea into results, we characterized every BioBrick as listed in [Parts](/Team%3AStockholm/Parts) and described in [Design](/Team%3AStockholm/Design). All of the bricks were transformed into TOP10 cells and tested for the following parameters:

* Growth curve/Toxicity of the constructs
* Fluorescent fingerprint using spectrophotometry and flow cytometry
* Protein expression using western blot

Here, you can find the most important findings for each BioBrick, and we urge you to follow the links found after each section for in-depth analysis.

![](https://static.igem.org/mediawiki/2019/e/e3/T--Stockholm--results-01.jpeg)

## The Switch Plasmid

The first part of our project revolves around creating the [**Switch Plasmid**](/Team%3AStockholm/results-switch-plasmid), a simulation of our inducible switch to force the P2 phage into the lysogenic cycle. For this to happen, we constructed two modules, the lysogenic [**C-protein Characterization Construct** (Lysogenic module)](/Team%3AStockholm/results-lysogenic-module) and the lytic [**Inducible cox characterization construct** (Lytic module)](/Team%3AStockholm/results-lytic-module). As we tested the lysogenic module, we saw no RFP expression through spectrophotometric analysis. Yet with western blot, we detected a constitutive C-protein expression, which confirmed that the bricks work as expected. As the lytic module was characterized, we saw arabinose-dependent toxicity which was not present in the control. Furthermore, no expression of neither GFP nor the cox-protein was found. When combined in the Switch Plasmid, we concluded from the western blot that there is a distinct expression of C-protein in non-induced samples and upon induction an increase in the cox expression, confirming that the switch works!

## The Model Plasmid

The second part of our project was the creation of a [**Model Plasmid**](/Team%3AStockholm/results-model-plasmid) to simulate the P2 genome and its reaction to the induction of the switch. We combined two modules; the [**Inducible C-protein construct**](/Team%3AStockholm/results-inducible-c-protein) and the [**Phage genome simulation module**](/Team%3AStockholm/results-phage-genome-simulation-module). The first module enabled careful control of the C-protein expression. western blot results showed that overexpression of C-protein was achieved at levels of induction above 0.4% arabinose. In line with our expectations, C-protein increased upon arabinose induction. Then, we created the second module that would mimic the phage switch genome. We showed that cox-protein is expressed on a stable level. However, we experienced issues in the detection of fluorescence.

## Discussion

When we discovered that fluorescence spectrophotometry could not detect fluorescence from our reporters, we began to investigate… We analyzed the sequences of our construct and saw that everything had been inserted, although, the RBS sequences were 4bp too short.

![](https://static.igem.org/mediawiki/2019/3/36/T--Stockholm--results-02.jpg)

### A new hope

We ordered a new **Switch Plasmid** construct containing the correct RBS sequences; furthermore, we ordered primers to correct for the RBS sequences for [BBa\_K3024004](http://parts.igem.org/Part%3ABBa_K3024004) and [BBa\_K3024011](http://parts.igem.org/Part%3ABBa_K3024011), two of our composite parts. With the new constructs, we again did not see an apparent increase in the fluorescence signaling. Considering that we still couldn't detect the fluorescence, we discussed whether some single cells might be expressing low levels of fluorescence. Johan Rockberg, our PI, helped us set up a flow cytometry experiment to measure the fluorescence from single cells within a population. For the **Switch Plasmid**, we noticed that upon arabinose induction, it expressed higher levels of RFP, which correlated with the results seen from the western blot results. Because we did not detect a significant shift in expression of RFP/GFP upon induction, we assume that the missing base pairs in the RBS were not the issue.

Maximillian Karlander, a Ph.D. student, recommended transforming our constructs in a different bacterial strain that is a more efficient expression strain. We transformed the **Switch Plasmid** in BL21 E.coli and analyzed the level of fluorescence in the ClariOstar plate reader using the previous protocol. There was no evident difference in neither expression nor growth of the two strains.

Another theory was that the degradation tag fused to the reporter genes caused a quick targeting and degradation of newly formed RFP/GFP, which would prevent detection. Interestingly, the maturation time for RFP is quite slow, meaning that although the protein is transcribed, it takes time before it emits a fluorescent signal.

### Planning ahead

To further investigate, we would perform RT-qPCR to assess whether there are detectable levels of the RFP/GFP mRNA transcripts. We aim to understand if the problem of expression originates from the translation or transcription. Ultimately, if there was a problem with the transcription, the cis- or tris-construct design should be considered as a non-functional concept!

On continuing the project, we would make all parts of the construct functional and further evaluate the protein expression using other characterization methods. We would also work on the integration of the envisioned sensing system with the **Switch Plasmid**. We would test the induction of bacteria in different pH concentrations similar to the gastric and intestinal fluids. Last but not least, we would aim to test the system with the P2 knockout phage.