iGEM 2019 — Team Purdue

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# Team:Purdue/Notebook

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[HOME](https://2019.igem.org/Team%3APurdue)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:Purdue/Experiments

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Team

[Team](https://2019.igem.org/Team%3APurdue/Team)
[Sponsors](https://2019.igem.org/Team%3APurdue/Sponsors)
[Attributions](https://2019.igem.org/Team%3APurdue/Attributions)
[Collaborations](https://2019.igem.org/Team%3APurdue/Collaborations)

Human Practices

[Human Practices](https://2019.igem.org/Team%3APurdue/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3APurdue/Public_Engagement)

Results

[Results](https://2019.igem.org/Team%3APurdue/Results)
[Parts](https://2019.igem.org/Team%3APurdue/Parts)

Project

[Description](https://2019.igem.org/Team%3APurdue/Description)
[Design](https://2019.igem.org/Team%3APurdue/Design)
[Experiments](https://2019.igem.org/Team%3APurdue/Experiments)
 [Protocols](https://2019.igem.org/Team%3APurdue/Documentation)
[Safety](https://2019.igem.org/Team%3APurdue/Safety)

Our summer project began with preliminary research on how chitin was produced in bacterial cells. When researching how bacterial cells produce chitin, we first had to decide what bacterial vectors we would be working with. We decided upon Pseudomonas fluorescens as the vector we were transforming into, and *Escheria coli* as the first chassis for the gene of interest. We selected *Pseudomonas* as our vector because this microbe is commonly found in soil and would be easy to integrate into its natural habitat near rice plants. *E. coli* was selected as the first organism to transform into because it is commonly used in synthetic biology, and its metabolic pathways are well-defined. We were able to determine that *E. coli* can produce chitin, but that is not a metabolic pathway that is normally utilized in the wildtype.

We also determined how chitin is utilized by *Magnaporthe oryzae*, and how plant immune systems work to recognize this product. Chitin is produced across both the outer membrane and inner membrane in bacteria, so we determined that we wouldn’t have to lyse the cells when confirming the product with the calcofluor white assay. The calcofluor white assay was used to confirm the presence of chitin- when calcofluor white binds to chitin, it fluoresces the color blue under a UV light.

Another main facet of our research was understanding the impact rice blast fungus has on rice crops worldwide. We aggregated data from many studies on the economic impact of rice blast fungus and were able to discover that rice blast fungus causes around 70 million dollars in damage yearly. Up to 30% of rice crops are damaged by this fungus every year, and this amount lost could feed around 60 million people. Since rice blast fungus has such a large and worldwide economic impact, this only solidified our decision to come up with some sort of counteracting measure.

All of the protocols that were utilized for our experiments can be viewed by clicking the red button below.

[Protocols](https://static.igem.org/mediawiki/2019/3/31/T--Purdue--Protocols.pdf)

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# Team:Purdue/Results

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Team

[Team](https://2019.igem.org/Team%3APurdue/Team)
[Sponsors](https://2019.igem.org/Team%3APurdue/Sponsors)
[Attributions](https://2019.igem.org/Team%3APurdue/Attributions)
[Collaborations](https://2019.igem.org/Team%3APurdue/Collaborations)

Human Practices

[Human Practices](https://2019.igem.org/Team%3APurdue/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3APurdue/Public_Engagement)

Results

[Results](https://2019.igem.org/Team%3APurdue/Results)
[Parts](https://2019.igem.org/Team%3APurdue/Parts)

Project

[Description](https://2019.igem.org/Team%3APurdue/Description)
[Design](https://2019.igem.org/Team%3APurdue/Design)
[Experiments](https://2019.igem.org/Team%3APurdue/Experiments)
 [Protocols](https://2019.igem.org/Team%3APurdue/Documentation)
[Safety](https://2019.igem.org/Team%3APurdue/Safety)

**PGEM:**
     Our plasmids were transformed into JM109 cells and ligated into the pGEM vector to determine if the gene of interest was inserted correctly. The bacteria were then plated onto an LB plate with X-gal, IPTG, and Ampicillin so that the colonies would grow to either be blue or white- the white colonies were selected from these plates for pure cultures to be made from. The presence of the gene product would then be later confirmed with an enzyme digest.

**Agarose Gel images:**
     Plasmids isolated from white colonies on the pGEM were digested with EcoR1 to confirm the presence of desired sequences in the pGEM backbone. Gel electrophoresis was performed to visualize the bands. If the band appeared in the correct location, then the miniprepped DNA was sent off for sequencing as the last confirmatory step.

**SDS Page:**
     An SDS Page gel was used to visualize and identify the protein molecules produced by Gold 1. The gel shown below confirmed the expression of the 20 kDa NodC protein with the SDS page

![](https://static.igem.org/mediawiki/2019/0/0d/T--Purdue--SDSpage.jpg )

**Calcofluor White Stain:**
     Calcofluor white is a stain that fluoresces blue when in the presence of chitin and UV light. As seen below, colonies with that contain a gene expressing chitin fluoresce blue much brighter than the wildtype strain. The calcofluor white assay was used to determine if chitin was being produced by our cells. Both a blue and a white colony were selected from the pGEM plate, and it was assumed that the blue colony would not have the gene inserted and the white colony would. Each colony was suspended in 200 microliters of PBS, and then 20 microliters of calcofluor white was added to the tube. The cells then sat for one minute, and then 40 microliters were pipetted onto a microscope slide. Once the glass coverslip was put on, the slide was analyzed under a fluorescent microscope. The cells were observed on a normal light setting and with UV light, and then an overlay image was created to compare those two views.

![](https://static.igem.org/mediawiki/2019/b/b8/T--Purdue--S3controlS4.jpg)

The figure above shows Pc with NodC\_Pf, a negative control, and PpsbA with NodC\_Pf. The negative control was used to show that the wildtype E. coli does not normally fluoresce. Pc with NodC\_Pf had almost all of its cells fluoresce blue, and PpsbA with NodC\_Pf had most of its cells fluoresce as well

![](https://static.igem.org/mediawiki/2019/8/80/T--Purdue--G1comparison.jpg )

The figure shown above shows NodC with an Anderson on the top and a negative control from the same plate on the bottom. The chitin product was shown to be produced since the cells fluoresce after being mixed with the calcofluor white assay. The control was used as a comparison to show how wildtype E. coli does not have most of its cells fluoresce.

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