iGEM 2018 — Team Purdue

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Purdue/Notebook

Bootstrap Example

[![](https://static.igem.org/mediawiki/2018/5/50/T--Purdue--Purdue_iGEM_Gold_Helix.png)](https://2018.igem.org/Team%3APurdue)

* [Overview](https://2018.igem.org/Team%3APurdue/Description)
  + [Problem](https://2018.igem.org/Team%3APurdue/Description)
  + [Solution](https://2018.igem.org/Team%3APurdue/Design)
  + [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)
* [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Integrated Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Survey](https://2018.igem.org/Team%3APurdue/Survey)
  + [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)
* [Project](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Genetic Construct](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
  + [Model](https://2018.igem.org/Team%3APurdue/Model)
  + [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
  + [Safety](https://2018.igem.org/Team%3APurdue/Safety)
  + [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
  + [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
  + [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)
* [Results](https://2018.igem.org/Team%3APurdue/Parts)
  + [Parts](https://2018.igem.org/Team%3APurdue/Parts)
  + [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)
* [Team](https://2018.igem.org/Team%3APurdue/Team)
  + [Biography](https://2018.igem.org/Team%3APurdue/Team)
  + [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

* Overview

+ [Problem](https://2018.igem.org/Team%3APurdue/Description)
+ [Solution](https://2018.igem.org/Team%3APurdue/Design)
+ [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)

* Human Practices

+ [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
+ [Survey](https://2018.igem.org/Team%3APurdue/Survey)
+ [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)

* Project

+ [Genetic Pathway](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
+ [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
+ [Model](https://2018.igem.org/Team%3APurdue/Model)
+ [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
+ [Safety](https://2018.igem.org/Team%3APurdue/Safety)
+ [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
+ [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
+ [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)

* Results

+ [Parts](https://2018.igem.org/Team%3APurdue/Parts)
+ [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)

* Team

+ [Biography](https://2018.igem.org/Team%3APurdue/Team)
+ [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

![](https://static.igem.org/mediawiki/2018/0/0f/T--Purdue--White.jpg)

# Week 1 (5/24/18)

Created GANTT chart, went over lab protocols, researched background information for yeast infections, discussed researchers and companies to reach out to for integrated human practices, made media, resuspended g blocks, created navigation and footer for wiki, purchased plane tickets, and reached out to companies for sponsorships.

# Week 2 (6/1/19)

Began cloning of gBlocks into E. coli, contacted planned parenthood, local hospitals, and a gynecologist, began Promega and Learning Beyond the Classroom grant, created wiki quiz and updated aesthetics, ordered lab supplies, met with colleges to discuss iGEM funding.

# Week 3 (6/7/18)

Used ampicillin plates to select for successfully cloned gBlocks, began model development using protein mass balance equation, scheduled meetings with professors on campus to discuss project, rough drafts for wiki content worked on, and began research on paper-based assay including discussions of design, type of paper, and characteristics.

# Week 4 (6/14/18)

Ran digests of gBlocks to confirm transformation, PCR failed for positive control development, furthered model development to account for speed of liquid falling through paper assay and rate of enzyme reaction, modeled capillary action with washburn equation and enzyme kinetics with michaelis-menten, discussed mechanisms of paper-based assay with professors on campus, created navigation bar on wiki, worked on questions for IRB survey, met with College of Engineering for funding request.

# Week 5 (6/19/18)

Successfully cloned most gBlocks into E. coli, discovered positive control development failed due to incorrect primers being ordered, contacted statistics department for preparation of IRB survey, began human subjects training, developed method to discover parameters for modeling, added navigational buttons to wiki, began training with professors for paper-based assay development.

# Week 6 (6/28/18)

Met with Purdue Research Foundation to discuss potential of getting patents for research, decided on parameters, paper, and design for paper-based assay, did restyling of wiki, learned how to use flex station for biological and paper-based assay.

# Week 7 (7/5/18)

Performed half of standardization experiments for interlab study, determined protein protein interaction constants for modeling, discovered error between simulation data and real parameter values, new positive control dimers continued to dimerize, casing PCR complications, met with girl scouts for potential human practices opportunity, applied design section added to wiki, and discussed paper-based assay shelf life and surfactant complications, as well as how to improve on cost, accuracy, durability, and speed.

# Week 8 (7/12/18)

Began photo scanner training for paper-based assay, began TMB biological assay development, restarted interlab study due to accidental freezing of microspheres, ran MCMC bayesian parameter estimation code, ran sensitivity analyses for parameters, contacted vice-provost for potential funding, brainstormed project acronyms, and discussed target audience for project.

# Week 9 (7/19/18)

Began aging experiments with TMB, characterized device using food colored water glucose solutions, whatman #1 chosen as paper-based assay paper, specifically 3mm thick due to ability to filter out red blood cells, projected costs ignoring reagents to roughly 8 cents, using plate reader we were able to quantify assay parameters with glucose solutions, adjusted parameters for developing positive control by changing annealing temperature of primers in PCR, completed all 3 calibrations for interlab study, and received Learning Beyond the Classroom Grant.

# Week 10 (7/26/18)

Created HRP calibration curve, discovered HRP is extremely reactive and will react in minutes if left in aerobic environment, needs peroxide to react, discovered red/orange intermediate is formed before fully oxidized, set experiment for adjusting TMB, HRP, and peroxide concentration to improve assay, discovered frb-fkbp interactions are rate-limited, used Markov Chain Monte Carlo to assess Bayesian Parameter Estimation, successfully cloned positive control into E.coli, completed interlab study, and ran escape room with DNA scouts on campus.

# Week 11 (8/2/18)

Sequenced positive control, proved successful transformation into E.coli, and transformed plasmid into yeast cells to be used in biological assay.

# Special Thanks

[to all those that made this possible](https://2018.igem.org/Team%3APurdue/Acknowledgement)
![](https://static.igem.org/mediawiki/2018/a/af/T--Purdue--purdue_icon.png)

# Follow Us On

[![](https://static.igem.org/mediawiki/2018/9/9f/T--Purdue--twitter.png)](https://twitter.com/purdueigem)

[![](https://static.igem.org/mediawiki/2018/7/74/T--Purdue--instagram.png)](https://www.instagram.com/purdueigem/)

[![](https://static.igem.org/mediawiki/2018/d/d5/T--Purdue--facebook.png)](https://www.facebook.com/PurdueiGEM/)

# Contact Us

igem@purdue.edu

Kevin Fitzgerald, B025

1203 W State St.

West Lafayette, IN 47906


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Purdue/Experiments

Bootstrap Example

[![](https://static.igem.org/mediawiki/2018/5/50/T--Purdue--Purdue_iGEM_Gold_Helix.png)](https://2018.igem.org/Team%3APurdue)

* [Overview](https://2018.igem.org/Team%3APurdue/Description)
  + [Problem](https://2018.igem.org/Team%3APurdue/Description)
  + [Solution](https://2018.igem.org/Team%3APurdue/Design)
  + [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)
* [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Integrated Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Survey](https://2018.igem.org/Team%3APurdue/Survey)
  + [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)
* [Project](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Genetic Construct](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
  + [Model](https://2018.igem.org/Team%3APurdue/Model)
  + [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
  + [Safety](https://2018.igem.org/Team%3APurdue/Safety)
  + [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
  + [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
  + [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)
* [Results](https://2018.igem.org/Team%3APurdue/Parts)
  + [Parts](https://2018.igem.org/Team%3APurdue/Parts)
  + [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)
* [Team](https://2018.igem.org/Team%3APurdue/Team)
  + [Biography](https://2018.igem.org/Team%3APurdue/Team)
  + [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

* Overview

+ [Problem](https://2018.igem.org/Team%3APurdue/Description)
+ [Solution](https://2018.igem.org/Team%3APurdue/Design)
+ [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)

* Human Practices

+ [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
+ [Survey](https://2018.igem.org/Team%3APurdue/Survey)
+ [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)

* Project

+ [Genetic Pathway](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
+ [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
+ [Model](https://2018.igem.org/Team%3APurdue/Model)
+ [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
+ [Safety](https://2018.igem.org/Team%3APurdue/Safety)
+ [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
+ [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
+ [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)

* Results

+ [Parts](https://2018.igem.org/Team%3APurdue/Parts)
+ [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)

* Team

+ [Biography](https://2018.igem.org/Team%3APurdue/Team)
+ [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

![](https://static.igem.org/mediawiki/2018/9/99/T--Purdue--drugs.jpg)

# Cloning Wild Type HRP

Part of the project involved the transformation of pFL-HRP into *Saccharomyces cerevisiae* strain BJ5464 with a Gal1 Promoter. The data we gathered suggested we were successful in this endeavor.

## Plasmid Sequencing

After confirming that we have a possible success, we extracted the shuttle vector DNA and submitted it for sequencing. The results of which can be seen below. gBlock 1 represents DNA with our composite part containing the gene for expressing full length HRP.

alt :

sequence.pdf

## PCR

After successful growth on the leucine dropout plates, 12 large colonies were chosen from one of the plates to test whether or not pFL-HRP was inserted into the DNA. The goal of this PCR experiment was to amplify part of gBlock 1, the composite part containing the full length HRP gene. The DNA part amplified in this experiment was expected to be 1395 base pairs long. 7 of the colonies showed around the expected base pair size of 1395.

![](https://static.igem.org/mediawiki/2018/6/6a/T--Purdue--HRPPCRGEL.png)

![](https://static.igem.org/mediawiki/2018/b/b8/T--Purdue--Yeast_Transformation_Plates.jpg)

# SDS-PAGE HRP Expression

## Protein expression using a protein gel

![](https://static.igem.org/mediawiki/2018/c/c7/T--Purdue--HRPproteinGel.jpeg)

A SDS-PAGE gel was used to determine the different sized proteins in 2 lysate samples with the goal of identifying expression of HRP in our construct. Protein samples were taken from yeast transformed with the plasmid pFL-HRP by lysing the cells and saving the lysate. When this yeast is grown in the presence of galactose, this yeast will express HRP. Any other media that does not contain galactose will inhibit the expression of HRP. To test expression of HRP, the yeast containing the plasmid pFL-HRP was grown in 1% galactose-containing media and the lysate from these colonies is labeled “pFL-HRP Gal 1%” in the image to the left. The second lysate sample was made from yeast containing the plasmid pFL-HRP as well. However, it was grown in YPD media that does not contain galactose. This sample is labeled “Negative Control” because this sample should not contain HRP in the lysate. The third sample is a positive control, which contains only pure HRP. After analyzing the gel, there is no definitive evidence that the pFL-HRP Gal 1% sample contains HRP.

# HRP Characterization

![](https://static.igem.org/mediawiki/2018/6/68/T--Purdue--LysateInhibition.png)

The simplest way to test for the presence of functional HRP is to mix reagents necessary for a colorimetric into the sample of interest; in our case, the sample is full-cell lysate and the reagents include TMB and H2O2. To prepare samples, 50mL cultures of our transformed yeast were inoculated in both galactose and glucose media. When initial tests failed to demonstrate HRP production in the induced sample we investigated potential causes. Using store-bought samples of HRP, we tested the colorimetric reaction at different concentrations of lysate. Using constant final concentrations of HRP, TMB, and H2O2 we discovered that an increase in lysate concentration corresponds to an apparent decrease in the extent of reaction. Presumably, something in the lysate inhibits the reaction from occurring. Our advisors indicated that native yeast proteins like catalase or superoxide dismutase could potentially consume the added H2O2 before HRP has the opportunity to react. With this information we added copper ions, an inhibitor to these proteins, to the cell lysates at a final concentration of 50mM in the hopes of witnessing an HRP-based color change. Although evidence suggests the ions were successful in inhibiting the native proteins, they also reacted with the lysate to produce a precipitate and an unexpected color change.

# Special Thanks

[to all those that made this possible](https://2018.igem.org/Team%3APurdue/Acknowledgement)
![](https://static.igem.org/mediawiki/2018/a/af/T--Purdue--purdue_icon.png)

# Follow Us On

[![](https://static.igem.org/mediawiki/2018/9/9f/T--Purdue--twitter.png)](https://twitter.com/purdueigem)

[![](https://static.igem.org/mediawiki/2018/7/74/T--Purdue--instagram.png)](https://www.instagram.com/purdueigem/)

[![](https://static.igem.org/mediawiki/2018/d/d5/T--Purdue--facebook.png)](https://www.facebook.com/PurdueiGEM/)

# Contact Us

igem@purdue.edu

Kevin Fitzgerald, B025

1203 W State St.

West Lafayette, IN 47906

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Purdue/Results

Bootstrap Example

[![](https://static.igem.org/mediawiki/2018/5/50/T--Purdue--Purdue_iGEM_Gold_Helix.png)](https://2018.igem.org/Team%3APurdue)

* [Overview](https://2018.igem.org/Team%3APurdue/Description)
  + [Problem](https://2018.igem.org/Team%3APurdue/Description)
  + [Solution](https://2018.igem.org/Team%3APurdue/Design)
  + [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)
* [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Integrated Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
  + [Survey](https://2018.igem.org/Team%3APurdue/Survey)
  + [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)
* [Project](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Genetic Construct](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
  + [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
  + [Model](https://2018.igem.org/Team%3APurdue/Model)
  + [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
  + [Safety](https://2018.igem.org/Team%3APurdue/Safety)
  + [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
  + [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
  + [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)
* [Results](https://2018.igem.org/Team%3APurdue/Parts)
  + [Parts](https://2018.igem.org/Team%3APurdue/Parts)
  + [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)
* [Team](https://2018.igem.org/Team%3APurdue/Team)
  + [Biography](https://2018.igem.org/Team%3APurdue/Team)
  + [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

* Overview

+ [Problem](https://2018.igem.org/Team%3APurdue/Description)
+ [Solution](https://2018.igem.org/Team%3APurdue/Design)
+ [Navigation](https://2018.igem.org/Team%3APurdue/Navpage)

* Human Practices

+ [Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
+ [Survey](https://2018.igem.org/Team%3APurdue/Survey)
+ [Collaboration](https://2018.igem.org/Team%3APurdue/Collaborations)

* Project

+ [Genetic Pathway](https://2018.igem.org/Team%3APurdue/Genetic_Pathway)
+ [Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
+ [Model](https://2018.igem.org/Team%3APurdue/Model)
+ [Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
+ [Safety](https://2018.igem.org/Team%3APurdue/Safety)
+ [Protocols](https://2018.igem.org/Team%3APurdue/Protocols)
+ [Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
+ [Interlab](https://2018.igem.org/Team%3APurdue/InterLab)

* Results

+ [Parts](https://2018.igem.org/Team%3APurdue/Parts)
+ [Judging Form](https://igem.org/2018_Judging_Form?team=Purdue)

* Team

+ [Biography](https://2018.igem.org/Team%3APurdue/Team)
+ [Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

# 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 be on part pages in the Registry.
* 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)

# Special Thanks

[to all those that made this possible](https://2018.igem.org/Team%3APurdue/Acknowledgement)
![](https://static.igem.org/mediawiki/2018/a/af/T--Purdue--purdue_icon.png)

# Follow Us On

[![](https://static.igem.org/mediawiki/2018/9/9f/T--Purdue--twitter.png)](https://twitter.com/purdueigem)

[![](https://static.igem.org/mediawiki/2018/7/74/T--Purdue--instagram.png)](https://www.instagram.com/purdueigem/)

[![](https://static.igem.org/mediawiki/2018/d/d5/T--Purdue--facebook.png)](https://www.facebook.com/PurdueiGEM/)

# Contact Us

igem@purdue.edu

Kevin Fitzgerald, B025

1203 W State St.

West Lafayette, IN 47906