iGEM 2017 — Team Minnesota

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# Team:Minnesota/Notebook

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MENU

[HOME](http://2017.igem.org/Team%3AMinnesota)

TEAM

[Team](http://2017.igem.org/Team%3AMinnesota/Team)
[Collaborations](http://2017.igem.org/Team%3AMinnesota/Collaborations)

PROJECT

[Description](http://2017.igem.org/Team%3AMinnesota/Description)
[Design](http://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](http://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](http://2017.igem.org/Team%3AMinnesota/Notebook)
[InterLab](http://2017.igem.org/Team%3AMinnesota/InterLab)
[Contribution](http://2017.igem.org/Team%3AMinnesota/Contribution)
[Model](http://2017.igem.org/Team%3AMinnesota/Model)
[Results](http://2017.igem.org/Team%3AMinnesota/Results)
[Demonstrate](http://2017.igem.org/Team%3AMinnesota/Demonstrate)
[Improve](http://2017.igem.org/Team%3AMinnesota/Improve)
[Attributions](http://2017.igem.org/Team%3AMinnesota/Attributions)

PARTS

[Parts](http://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](http://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](http://2017.igem.org/Team%3AMinnesota/Composite_Part)
[Part Collection](http://2017.igem.org/Team%3AMinnesota/Part_Collection)

[SAFETY](http://2017.igem.org/Team%3AMinnesota/Safety)

HUMAN PRACTICES

[Silver HP](http://2017.igem.org/Team%3AMinnesota/HP/Silver)
[Integrated and Gold](http://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Public Engagement](http://2017.igem.org/Team%3AMinnesota/Engagement)

AWARDS

[Applied Design](http://2017.igem.org/Team%3AMinnesota/Applied_Design)
[Entrepreneurship](http://2017.igem.org/Team%3AMinnesota/Entrepreneurship)
[Hardware](http://2017.igem.org/Team%3AMinnesota/Hardware)
[Measurement](http://2017.igem.org/Team%3AMinnesota/Measurement)
[Model](http://2017.igem.org/Team%3AMinnesota/Model)
[Plant](http://2017.igem.org/Team%3AMinnesota/Plant)
[Software](http://2017.igem.org/Team%3AMinnesota/Software)

[JUDGING FORM](http://igem.org/2017_Judging_Form?team=Minnesota)

# Minnesota

[Home](http://2017.igem.org/Team%3AMinnesota)

Team

[Team](http://2017.igem.org/Team%3AMinnesota/Team)
 [Collaborations](http://2017.igem.org/Team%3AMinnesota/Collaborations)

Project

[Description](http://2017.igem.org/Team%3AMinnesota/Description)
[Design](http://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](http://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](http://2017.igem.org/Team%3AMinnesota/Notebook)
[Model](http://2017.igem.org/Team%3AMinnesota/Model)
[Results](http://2017.igem.org/Team%3AMinnesota/Results)
[Attributions](http://2017.igem.org/Team%3AMinnesota/Attributions)

Parts

[Parts](http://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](http://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](http://2017.igem.org/Team%3AMinnesota/Composite_Part)

[Safety](http://2017.igem.org/Team%3AMinnesota/Safety)

Human Practices

[Integrated and Gold](http://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Documentary](http://2017.igem.org/Team%3AMinnesota/Engagement)

MINNESOTA

Notebook Links

 [Lab Notebook](https://docs.google.com/document/d/1PLJDq0P6ESC1e587JNOkKZW6TqT_pR2JM-QgKDY_g0k/edit)

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# Team:Minnesota/Experiments

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3AMinnesota)

MENU

[HOME](https://2017.igem.org/Team%3AMinnesota)

TEAM

[Team](https://2017.igem.org/Team%3AMinnesota/Team)
[Collaborations](https://2017.igem.org/Team%3AMinnesota/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3AMinnesota/Description)
[Design](https://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](https://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](https://2017.igem.org/Team%3AMinnesota/Notebook)
[InterLab](https://2017.igem.org/Team%3AMinnesota/InterLab)
[Contribution](https://2017.igem.org/Team%3AMinnesota/Contribution)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Results](https://2017.igem.org/Team%3AMinnesota/Results)
[Demonstrate](https://2017.igem.org/Team%3AMinnesota/Demonstrate)
[Improve](https://2017.igem.org/Team%3AMinnesota/Improve)
[Attributions](https://2017.igem.org/Team%3AMinnesota/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](https://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AMinnesota/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3AMinnesota/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AMinnesota/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3AMinnesota/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AMinnesota/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3AMinnesota/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3AMinnesota/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3AMinnesota/Hardware)
[Measurement](https://2017.igem.org/Team%3AMinnesota/Measurement)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Plant](https://2017.igem.org/Team%3AMinnesota/Plant)
[Software](https://2017.igem.org/Team%3AMinnesota/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Minnesota)

# Minnesota

[Home](https://2017.igem.org/Team%3AMinnesota)

Team

[Team](https://2017.igem.org/Team%3AMinnesota/Team)
 [Collaborations](https://2017.igem.org/Team%3AMinnesota/Collaborations)

Project

[Description](https://2017.igem.org/Team%3AMinnesota/Description)
[Design](https://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](https://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](https://2017.igem.org/Team%3AMinnesota/Notebook)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Results](https://2017.igem.org/Team%3AMinnesota/Results)
[Attributions](https://2017.igem.org/Team%3AMinnesota/Attributions)

Parts

[Parts](https://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](https://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AMinnesota/Composite_Part)

[Safety](https://2017.igem.org/Team%3AMinnesota/Safety)

Human Practices

[Integrated and Gold](https://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Documentary](https://2017.igem.org/Team%3AMinnesota/Engagement)

MINNESOTA

**Experiment 1: Creation of the FitD Plasmid.**
The E. coli will be transformed with the PBAD18 plasmid and grown in the absence of arabinose to ensure that transcription of the plasmid genes, including cytolysin FitD cannot occur, thus limiting the chance of prematurely killing E. coli cells in the event that cytolysin FitD is toxic to the cells. Following transformation, the PBAD18 E. coli will be grown in the presence of arabinose, thus allowing transcription of the genes on the PBAD18 plasmid, including FitD. If the E. coli cells survive growth in arabinose, a coomassie gel will be run and the amount of cytolysin FitD will be quantified.

 **Experiment 2: Creation of Purine Auxotrophy System**
Design and synthesize the PurA plasmid. Transform E. coli with plasmid. To test the delayed auxotrophy system, the UMN iGEM team will culture the engineered E. coli in a series of culture tubes without IPTG. At various time points, the turbidity of the culture will be measured using spectroscopy in order to quantify the number of viable cells. Cultures will also be plated out and CFU/ml will be determined. If the delayed auxotrophy system works, it is expected that the number of viable cells will decrease in relation to time.

**Experiment 3: Co-Transform Purine and FitD Plasmid**
The two plasmids will be co-transformed into E. coli. The E. coli will then be streaked on media containing kanamycin and ampicillin in order to select for the cells that contain both plasmids. To test the efficiency of the co-transfection, an additional plasmid will be designed that has both of the expression systems on it. These two systems will be tested to see which is most efficient and which has the best control for release in the environment.

**Experiment 4: LD50 of FitD system**
The Minnesota Aquatic Invasive Species Research Center (MAISRC), which works in partnership with the University of Minnesota, has agreed to provide us with zebra mussels so an experiment to determine LD50 can be run on the engineered E. coli.

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# Team:Minnesota/Results

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3AMinnesota)

MENU

[HOME](https://2017.igem.org/Team%3AMinnesota)

TEAM

[Team](https://2017.igem.org/Team%3AMinnesota/Team)
[Collaborations](https://2017.igem.org/Team%3AMinnesota/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3AMinnesota/Description)
[Design](https://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](https://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](https://2017.igem.org/Team%3AMinnesota/Notebook)
[InterLab](https://2017.igem.org/Team%3AMinnesota/InterLab)
[Contribution](https://2017.igem.org/Team%3AMinnesota/Contribution)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Results](https://2017.igem.org/Team%3AMinnesota/Results)
[Demonstrate](https://2017.igem.org/Team%3AMinnesota/Demonstrate)
[Improve](https://2017.igem.org/Team%3AMinnesota/Improve)
[Attributions](https://2017.igem.org/Team%3AMinnesota/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](https://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AMinnesota/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3AMinnesota/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AMinnesota/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3AMinnesota/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AMinnesota/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3AMinnesota/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3AMinnesota/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3AMinnesota/Hardware)
[Measurement](https://2017.igem.org/Team%3AMinnesota/Measurement)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Plant](https://2017.igem.org/Team%3AMinnesota/Plant)
[Software](https://2017.igem.org/Team%3AMinnesota/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Minnesota)

# Minnesota

[Home](https://2017.igem.org/Team%3AMinnesota)

Team

[Team](https://2017.igem.org/Team%3AMinnesota/Team)
 [Collaborations](https://2017.igem.org/Team%3AMinnesota/Collaborations)

Project

[Description](https://2017.igem.org/Team%3AMinnesota/Description)
[Design](https://2017.igem.org/Team%3AMinnesota/Design)
[Experiments](https://2017.igem.org/Team%3AMinnesota/Experiments)
[Notebook](https://2017.igem.org/Team%3AMinnesota/Notebook)
[Model](https://2017.igem.org/Team%3AMinnesota/Model)
[Results](https://2017.igem.org/Team%3AMinnesota/Results)
[Attributions](https://2017.igem.org/Team%3AMinnesota/Attributions)

Parts

[Parts](https://2017.igem.org/Team%3AMinnesota/Parts)
[Basic Parts](https://2017.igem.org/Team%3AMinnesota/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AMinnesota/Composite_Part)

[Safety](https://2017.igem.org/Team%3AMinnesota/Safety)

Human Practices

[Integrated and Gold](https://2017.igem.org/Team%3AMinnesota/HP/Gold_Integrated)
[Documentary](https://2017.igem.org/Team%3AMinnesota/Engagement)

MINNESOTA

We were able to successfully create the PurA part, BBa\_K2375000. Additionally, we were able to codon optimize mCherry BBa\_J06504 by removing the internal Ribosomal Binding Site. This part was called BBa\_K2375001.

We spent a large majority of our time trying to optimize and create the FitD plasmid system. The Gibson’s Assembly failed multiple times due to nuclease contamination and simply the size of the gene that we were trying to create.

Additionally, we participated in the Interlab study, but we were unable to successfully transform the plasmids because we were given a chloramphenicol resistant E. coli strain.

However, upon beginning to work on our control systems, we had promising results, and we were able to create the PurA gene for use in our control systems.

Further work on this subject could be to continue creating the Purine Auxotropy system as well as experimenting with other auxotrophy and biocontrol systems.

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