iGEM 2019 — Team WLC-Milwaukee

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# Team:WLC-Milwaukee/Notebook

PLACEHOLDER

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2019/7/7e/T--WLC-Milwaukee--logo.png "WLC-Milwaukee iGEM Home")](/Team%3AWLC-Milwaukee)

[PROJECT](/Team%3AWLC-Milwaukee/Description)

[Overview](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
[System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
[Wet Lab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
[Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
[Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

[DOCUMENTATION](%5CTeam%3AWLC-Milwaukee%5CJudging)

[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
[Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

[HUMAN PRACTICES](%5CTeam%3AWLC-Milwaukee%5CHP)

[Overview](%5CTeam%3AWLC-Milwaukee%5CHP)
[Investigations and Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

[TEAM](%5CTeam%3AWLC-Milwaukee%5CTeam)

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2019/7/7e/T--WLC-Milwaukee--logo.png "WLC-Milwaukee iGEM Home")
WLC-Milwaukee iGEM](/Team%3AWLC-Milwaukee)

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PROJECT

[Overview](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
[System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
[Wetlab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
[Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
[Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

*developer\_board*
DOCUMENTATION

[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
[Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

*nature\_people*
HUMAN PRACTICES

[Overview](%5CTeam%3AWLC-Milwaukee%5CHP)
[Investigations and Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

*people*
TEAM

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

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WLC iGEM 2018 | Timeline

![](https://static.igem.org/mediawiki/2019/1/12/T--WLC-Milwaukee--TIMELINE.JPG)

# Project Timeline

#### November 2018

![Image](https://static.igem.org/mediawiki/2019/a/ae/T--WLC-Milwaukee--TIMELINE1.png)

---

Brainstorming new projects, coming up with human practices ideas, and getting acquainted with who will be doing what! Hanging out, decompressing after the 2018 jamboree, and getting ready for winter break.

#### February 4, 2019

---

Began lab protocol and safety training for the project. Alyssa taught basic pipetting and gel electrophoresis to Ben, Connor, and Emma.

#### March 30, 2019

---

The iGEM team does a service project for WLC! We cleaned every lab in our science building, Generac Hall. It was an excellent bonding experience and helped the professors immensely.

#### June 9, 2019

![Image](https://static.igem.org/mediawiki/2019/0/01/T--WLC-Milwaukee--TIMELINE3.png)

---

Ben and Dr. Werner meet to discuss research. In the next few days they decide on a general plan to create the biosensor.

#### July 9, 2019

---

Achieved successful clones of pbrR, T7, and BSMT1.

#### August 6, 2019

![Image](https://static.igem.org/mediawiki/2019/f/f0/T--WLC-Milwaukee--TIMELINE6.png)

---

Achieved successful clones of the entire system! All genes, prbR, pbrT, T7 promoter, and BSMT1 were successfully cloned and transformed into E. coli. We verified this by sequencing and gel electrophoresis.

#### October 5, 2019

---

Performed a western blot that turned showed some results, however proteins did not appear in the correct spots.

#### October 30, 2019

---

Will travel to Boston, present our research, and have a great time at the Jamboree! Despite less than desirable results.

#### January 22, 2019

![Image](https://static.igem.org/mediawiki/2019/7/79/T--WLC-Milwaukee--TIMELINE2.png)

---

Winter Org Smorg! Every semester at WLC, the campus hosts a smorgasbord of student lead organizations. The iGEM team was there in full force, recruiting new members and spreading awareness about lead contamination.

#### March 2019

---

Background research on lead contamination begins! Ben, Connor, and many other team members compile a large document filled with information about lead contamination and the symptoms of lead poisoning.

#### May 4, 2019

---

Members of the iGEM team help at the Milwaukee Undergraduate Biological Research Conference hosted by WLC. Specifically, team members helped with registration and logistical support, while getting to learn about all the exciting undergraduate research happening in the Milwaukee area.

#### June 20, 2019

![Image](https://static.igem.org/mediawiki/2019/e/e0/T--WLC-Milwaukee--TIMELINE5.png)

---

After all the genes have arrived at WLC, Ben and Dr. Werner begin research. They began by confirming each gene and plasmid was the correct length.

#### July 10, 2019

---

Ran our first experiment! Added our transformed E. coli to a sample of water with a known concentration of lead, and then added a small amount of salicylate. Unfortunately, we did not smell anything. We presume that pbrT was needed for effective function.

#### September 27, 2019

---

Began western blots to test for protein expression.

#### October 11, 2019

![Image](https://static.igem.org/mediawiki/2019/3/39/T--WLC-Milwaukee--TIMELINE7.png)

---

Performed another western blot, after tweaking antibodies to hopefully show the correct proteins in the correct spots. Unfortunately, the proteins that showed on the blots were of the incorrect lengths, leading us to conclude that no proteins are being produced by our system.

* [Project](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
* [System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
* [Wet Lab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
* [Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
* [Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

* [Documentation](%5CTeam%3AWLC-Milwaukee%5CNotebook)
* [Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
* [Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
* [Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

* [Human Practices](%5CTeam%3AWLC-Milwaukee%5CHP)
* [Investigations & Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
* [Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
* [Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

* [Team](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

![Wisconsin Lutheran College](https://static.igem.org/mediawiki/2018/7/79/T--WLC-Milwaukee--WLC.png)


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# Team:WLC-Milwaukee/Experiments

PLACEHOLDER

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2019/7/7e/T--WLC-Milwaukee--logo.png "WLC-Milwaukee iGEM Home")](/Team%3AWLC-Milwaukee)

[PROJECT](/Team%3AWLC-Milwaukee/Description)

[Overview](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
[System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
[Wet Lab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
[Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
[Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

[DOCUMENTATION](%5CTeam%3AWLC-Milwaukee%5CJudging)

[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
[Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

[HUMAN PRACTICES](%5CTeam%3AWLC-Milwaukee%5CHP)

[Overview](%5CTeam%3AWLC-Milwaukee%5CHP)
[Investigations and Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

[TEAM](%5CTeam%3AWLC-Milwaukee%5CTeam)

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2019/7/7e/T--WLC-Milwaukee--logo.png "WLC-Milwaukee iGEM Home")
WLC-Milwaukee iGEM](/Team%3AWLC-Milwaukee)

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PROJECT

[Overview](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
[System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
[Wetlab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
[Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
[Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

*developer\_board*
DOCUMENTATION

[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
[Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

*nature\_people*
HUMAN PRACTICES

[Overview](%5CTeam%3AWLC-Milwaukee%5CHP)
[Investigations and Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

*people*
TEAM

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

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WLC iGEM 2019 | Wetlab

# Wet Lab

![](https://static.igem.org/mediawiki/2019/0/00/T--WLC-Milwaukee--WETLAB.JPG)

WLC-Milwaukee’s wet lab work was done primarily in the summer, with a few lingering experiments being completed during the school year, into October. We focused primarily on designing a feasible and functional engineered E. Coli for use in sensing lead content in a sample of water. After deciding to pursue a test kit based around a sense of smell, the wet lab team began searching for viable parts to use.

We decided to use genes from the heavy metal resistant bacteria Cupriavidus metallidurans. Specifically, we chose this organism because of its inducible promoter and transport protein, which are sensitive to lead. We utilized this promoter, pbrR, as the basis for our project. When lead binds to the promoter, transcription is initiated for the genes attached to the promoter. Additionally, we used the gene pbrT, which ensured the uptake of lead by the cell. This provides a simple way for odor to be produced when the promoter is exposed to lead, given the gene downstream from the promoter codes for a type of odor.

This leads to the selection of a gene that will produce some sort of odor. We selected the gene benzoate/salicylate carboxyl methyltransferase, or BSMT1 (BBa\_J45004). This gene creates the enzyme methyltransferase, which converts salicylate into methylsalycylate, the chemical that has a wintergreen odor. By adding this gene adjacent to the lead inducible promoter, the assembled machine will produce wintergreen odor when exposed to lead.

Our wet lab team met with Dr. John Werner early in the summer to begin lab work. We began by ligating our desired genes into the psb1c3 plasmid and moved this plasmid into an E. Coli strain by electroporation. Specifically, we inserted BSMT1, pbrT, pbrR, the pbrABCD operon promoter, and the T7 promoter into the plasmid. We verified this with DNA gel electrophoresis as well as sending our clones for sequencing.

Specifically, the function of our system is as follows.

1. The pbrT gene produces the pbrT protein, which brings lead into the E. Coli cell.
2. Both the pbrR and pbrT genes are under the control of the T7 promoter, a promoter from the T7 bacteriophage.
3. This T7 promoter constantly expresses the pbrT and pbrR genes, which in turn constantly produce the pbrT and pbrR proteins.
4. Two pbrR proteins join to form a dimer.
5. Lead binds to this pbrR dimer, and the pbrR protein then binds to the pbrABCD promoter.
6. This promoter expresses the BSMT1 gene, which produces the enzyme that converts salicylate (salicylic acid) into methyl salicylate.
7. This methyl salicylate has a wintergreen odor; therefore wintergreen odor should only be detected when lead is present.

![](https://static.igem.org/mediawiki/2019/0/08/T--WLC-Milwaukee--WETLAB1.png)

This summer, the wet lab team also did work with previous parts from WLC-Milwaukee (BBa\_K1900000 and BBa\_K 1900001). In this work, the tolC genes from Klebsiella pneumoniae and Serratia marcescens were cloned into a plasmid containing the pTrc promoter. This plasmid was then transformed into an E. coli strain that lacked a chromosomal copy of tolC. We then performed disk sensitivity assays to determine how well the S. marcescens and K. pneumoniae TolC protein could perform in an E. Coli cell. Please see the results section for more information on these experiments.

[3] <https://www.uniprot.org/uniprot/Q58AJ4>

[4] <https://www.uniprot.org/uniprot/Q58AJ4>

* [Project](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Background](%5CTeam%3AWLC-Milwaukee%5CBackground)
* [System Design](%5CTeam%3AWLC-Milwaukee%5CApplied_Design)
* [Wet Lab](%5CTeam%3AWLC-Milwaukee%5CExperiments)
* [Results](%5CTeam%3AWLC-Milwaukee%5CDemonstrate)
* [Parts](%5CTeam%3AWLC-Milwaukee%5CImprove)

* [Documentation](%5CTeam%3AWLC-Milwaukee%5CNotebook)
* [Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
* [Timeline](%5CTeam%3AWLC-Milwaukee%5CNotebook)
* [Judging](%5CTeam%3AWLC-Milwaukee%5CJudging)

* [Human Practices](%5CTeam%3AWLC-Milwaukee%5CHP)
* [Investigations & Impact](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
* [Outreach](%5CTeam%3AWLC-Milwaukee%5CPublic_Engagement)
* [Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)

* [Team](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)

![Wisconsin Lutheran College](https://static.igem.org/mediawiki/2018/7/79/T--WLC-Milwaukee--WLC.png)

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# Team:WLC-Milwaukee/Results

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[HOME](https://2019.igem.org/Team%3AWLC-Milwaukee)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# Results

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

### What should this page contain?

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

### Describe what your results mean

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

### Project Achievements

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

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

### Inspiration

See how other teams presented their results.

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