iGEM 2019 — Team Auburn_Alabama

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# Team:Auburn Alabama/Notebook

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TEAM

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

PROJECT

[Description](https://2019.igem.org/Team%3AAuburn_Alabama/Description)
[Design](https://2019.igem.org/Team%3AAuburn_Alabama/Design)
[Experiments](https://2019.igem.org/Team%3AAuburn_Alabama/Experiments)
[Results](https://2019.igem.org/Team%3AAuburn_Alabama/Results)
[Attributions](https://2019.igem.org/Team%3AAuburn_Alabama/Attributions)

[SAFETY](https://2019.igem.org/Team%3AAuburn_Alabama/Safety)
[HUMAN PRACTICES](https://2019.igem.org/Team%3AAuburn_Alabama/Human_Practices)
[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Auburn_Alabama)

# Notebook

January 18th - first meeting to discuss our possible project idea
-we decided to focus on detecting possible harmful substances in makeup
-we assigned a possible substance for us to detect to each member to do further research on
-lead- Sarah, aluminum- Chase, sulfate- Andrew, paraben- Amy and Jaeyoung, Phtalates- Michael, Triclosan – Scott
January 24, 2019- second meeting
January 28th- looked at Avon laboratories, ARKRAY inc, and Siemens healthcare as practitioners of cell transformation stuff and synthetic biology (Amy)
February 19th – researched into cell free protein synthesis
March 8th – gave everyone an excerpt from the Genetic Engineering Book to read (pages 24-37)
March 18th – planned next weeks practice transformation experiment with kit
KIT:
1x DNA Playground
1x Zero to Genetic Engineering Hero book
4x Canvas Kits
1x Group Engineer-it Kit (enough for 8 genetic engineering experiments - you can sub in your own DNA plasmids)
2x Plate Extract-it Kits4
March 22nd- pour plates
March 26th- practice transformation procedure with kit
March 27th- checked plates- two worked two failed
April 2nd- looked into other teams projects
(“Here are a few of last year’s high school teams and their projects:
Bioremediation of biofilm
https://2018.igem.org/Team:US\_AFRL\_CarrollHS
Inexpensive diagnostics kit to detect AND predict cholera outbreaks
https://2018.igem.org/Team:Lambert\_GA
Visual detection of excess fluoride in water
https://2018.igem.org/Team:East\_Chapel\_Hill
HIV-1 diagnostics kits for infants in developing nations
https://2018.igem.org/Team:Austin\_LASA
Arsenic detector
https://2018.igem.org/Team:Tacoma\_RAINmakers
Getting E. coli to light up LIGHTBULBS
https://2018.igem.org/Team:Pittsburgh\_CSL
Trying to cure Huntington’s disease
https://2018.igem.org/Team:New\_York\_City")
April 5th- discussed the other teams projects and what we could realistically do
April 9th- discussed our wiki and created bios, gave Alice pictures for our IGEM avatars
-looked into metal sensitive promoters that we could use in our plasmid, we were each supposed to look one up for the next meeting (in stock in bio brick registry)
April 16th- we talked about the promoters we found and now we were each supposed to find a reporter protein in the bio brick registry (in stock)
April 23rd – discussed reporters we found + Cowen Erwin joins
April 26th- discussed picking our RBS and whether we were going to focus on cobalt or mercury promoter; we decided on the cobalt promoter because it also could detect nickel but the team had not characterized it on the IGEM wiki
April 30th-
Began planning our summer and what we were going to do
May 3rd-
We discussed how we would do the transformation experiment with our individual parts (this was before we knew we could send in the plasmid pairs to companies to have them synthesized for us)
Also figured out that the RBS came with the kits IGEM sends us)
May 10th- Our logo is finished by Alice
May 30th- we discussed our summer plans and when we were going to do the transformation- mainly discussed schedules
June 5th- Discussed our transformation and finalized our schedules for the summer
June 7th- delegated duties to team members for the short term
-1. Safety form
-2.project description and inspiration (Andrew and Michael)
-3. IGEM wiki- Chase and Alice
-4. Human practices- Amy, Cady, Cowen, Jae
-5. Safety- Scott
June 11th- Skype with University of Alabama team
-rediscussed the order and method of our transformation and re-discussed our plans, talked about our progress
June 19th- was assigned our mentor
June 24th - Sarah completed the draft of the Safety Form, and Michael and Andrew completed the Project Description and Inspiration to our wiki.
July 1st - found out that plasmid construction is very hard as you lose yield in every step; so made IDT account and started designing our plasmid.
July 22nd
-ordered fully constructed plasmids from IDT
August 15th - received the plasmids, one with chromoprotein and another with fluorescnce that are sequence verified, that we ordered. Members gathered and discussed about transformation and characterizing.
September 1st - ordered nickel acetate powder
September 3rd - got our plasmids but we didn’t have IDTE buffer to resuspend them so planned to use distilled water for our transformation.
September 9th - Sarah worked on our wiki’s abstract.
September 11st– first trial for transformation
September 18th - found out that transformation did not work, so started second trial for transformation
September 19th - found out that transformation did not work, so started third trial for transformation
September 20th - found out that transformation did work
September 30th - tried testing with nickel solution to see if the transformation worked to change color. Found out that the color did not change.
October 5th – Tried to test again in nickel
October 6th - found out that the gene may be mutated?
October 12th - meeting at the public library to grind out wiki
-we assigned different sections of wiki pages to do
-Sarah – Project Design, Safety
-Scott – Safety, Notebook
-Michael - Notebook


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# Team:Auburn Alabama/Experiments

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PROJECT

[Description](https://2019.igem.org/Team%3AAuburn_Alabama/Description)
[Design](https://2019.igem.org/Team%3AAuburn_Alabama/Design)
[Experiments](https://2019.igem.org/Team%3AAuburn_Alabama/Experiments)
[Results](https://2019.igem.org/Team%3AAuburn_Alabama/Results)
[Attributions](https://2019.igem.org/Team%3AAuburn_Alabama/Attributions)

[SAFETY](https://2019.igem.org/Team%3AAuburn_Alabama/Safety)
[HUMAN PRACTICES](https://2019.igem.org/Team%3AAuburn_Alabama/Human_Practices)
[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Auburn_Alabama)

# Nickel “Kirby Bauer” Experiments

1. Inoculate transformed cells in LB broth, and grow overnight

2. Spread 150µl of cell culture on LB agar plate

3. Place an autoclave sterilized filter disk on plate

4. Add 10µl of nickel acetate solution

5. Incubate overnight

After we obtained our transformed cells, we performed this experiment to test our promoter for nickel sensitivity. This experiment was done with the cells containing the plasmid with prcn and the control cells containing a plasmid without prcn. Also, we performed the experiment at various concentrations of nickel acetate solution (5e8 ng/ml, 5e7 ng/ml, 5e6 ng/ml, 5e5 ng/ml, 50000 ng/ml, 5000 ng/ml, 500ng/ml, 50ng/ml, 5ng/ml, 0.5ng/ml).

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# Team:Auburn Alabama/Results

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TEAM

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PROJECT

[Description](https://2019.igem.org/Team%3AAuburn_Alabama/Description)
[Design](https://2019.igem.org/Team%3AAuburn_Alabama/Design)
[Experiments](https://2019.igem.org/Team%3AAuburn_Alabama/Experiments)
[Results](https://2019.igem.org/Team%3AAuburn_Alabama/Results)
[Attributions](https://2019.igem.org/Team%3AAuburn_Alabama/Attributions)

[SAFETY](https://2019.igem.org/Team%3AAuburn_Alabama/Safety)
[HUMAN PRACTICES](https://2019.igem.org/Team%3AAuburn_Alabama/Human_Practices)
[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Auburn_Alabama)

# Results

## Results of the Nickel “Kirby Bauer” Experiments

We found that at 5e8 ng/ml and 5e7 ng/ml of nickel acetate, most of surrounding cells died and a zone of inhibition formed. However, at all lower concentrations, nickel did not inhibit the growth of the cells on the plate.
Even after extended periods in the incubator, none of our transformed cells expressed the cjblue protein in nickel. Blue color was not present.

## Analysis

We did not have the resources to get our plasmids Sanger sequences, so we are unsure if there may have been a mutation in the gene. Although we cannot know for sure, the presence of a mutation is a possible reason why our cells did not express the blue chromoprotein.
A less likely possibility is that the gene may only express the blue chromoprotein at lethal concentrations of nickel. The promoter may only be responsive to concentrations of nickel that kill the cell.