iGEM 2017 — Team UGA-Georgia

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# Team:UGA-Georgia/Notebook

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* ![notebook](https://static.igem.org/mediawiki/2017/6/68/UGA-team.svg)

  Team

  [Team](https://2017.igem.org/Team%3AUGA-Georgia/Team)
  [Collaborators](https://2017.igem.org/Team%3AUGA-Georgia/Collaborators)
  [Attributions](https://2017.igem.org/Team%3AUGA-Georgia/Attributions)
* ![beaker](https://static.igem.org/mediawiki/2017/5/5c/UGA-beaker.svg)

  Project

  [Description](https://2017.igem.org/Team%3AUGA-Georgia#description)
  [Design](https://2017.igem.org/Team%3AUGA-Georgia/Design)

  [InterLab](https://2017.igem.org/Team%3AUGA-Georgia/InterLab)
  [Results](https://2017.igem.org/Team%3AUGA-Georgia/Results)
* [![notebook](https://static.igem.org/mediawiki/2017/d/df/UGA-notebook.svg)

  Notebook](https://2017.igem.org/Team%3AUGA-Georgia/Notebook)
* [![notebook](https://static.igem.org/mediawiki/2017/4/46/UGA-parts.svg)

  Parts](https://2017.igem.org/Team%3AUGA-Georgia/Parts)
* [![notebook](https://static.igem.org/mediawiki/2017/5/55/UGA-safety.svg)

  Safety](https://2017.igem.org/Team%3AUGA-Georgia/Safety)
* ![notebook](https://static.igem.org/mediawiki/2017/8/85/UGA-human-practices.svg)

  Human Practices

  [Public Engagement](https://2017.igem.org/Team%3AUGA-Georgia/Public_Engagement)
  [Survey](https://2017.igem.org/Team%3AUGA-Georgia/Survey)
* ![notebook](https://static.igem.org/mediawiki/2017/e/e3/UGA-achievements.svg)

  Achievements

  [Bronze](https://2017.igem.org/Team%3AUGA-Georgia/Bronze)
  [Silver](https://2017.igem.org/Team%3AUGA-Georgia/Silver)

* [Judging Form](https://igem.org/2017_Judging_Form?team=UGA-Georgia)
* [Wiki Design by: Logan Jahnke](http://loganjahnke.com)
* [University of Georgia iGem Team 2017](https://igem.org/2017_Judging_Form?team=UGA-Georgia)

# Notebook

# MAY

### Week 1

Made plasmids and prepared to test our constructs

Made LB broth and ampicillin agar plates

Prepared for transformation, plating, and extraction

### Week 2

Determine if the insert is present within the transformed expressor plasmid

Did not see an insert

### Week 3

Determine if we need to take any steps to visualize the GFP: UV light?

Prepared bacterial stocks of parent plasmids transformation completed

Begin examining the literature for aflatoxin detection strategies using the concentration of aflatoxin most prevalent
in nature

### Week 4

Digestions, gels, and gel purifications

Ligations (for both repressor and expressor)

Ligated plasmids transformed into DH3α cells

Minipreps of parent plasmids from iGEM were performed protocol is in the miniprep box.

# JUNE

### Week 1

Digestions, gels, gel purifications, and ligations

Ligated plasmids were transformed into DH3α cells

Minipreps of parent plasmids from iGEM were performed via protocol in the miniprep box

### Week 2

Gel Purification of 4 Vector digestion: each vector digested with EcoR1 and Pst1 +/- CIP

Column Purification of Insert digestions EcoR1 and Pst1

Ligations were performed and cells were transformed with litigation mixture

No GFP expression seen from our expressor

Double transformations done using expressor and repressor obtained from minipreps; double antibiotic (A & C) plates
were used for selection.

### Week 3

Updated sequences for expressor, repressor, expressor 3’ Truncated by 9 bp, Expressor 5’ Truncated by 9 bp, and expressor
with aptamer.

### Week 4

Picked and inoculated fluorescent colonies from the E31 streak plate. Picked and inoculated some fluorescent and
non-fluorescent colonies from the E3 and R spread plate.

# JULY

### Week 1

Extracted plasmids from previous week’s samples, digested appropriately, and visualized on a gel.

### Week 2

Met with PI about protocols for working with aflatoxin

### Week 3

Digestion of double E3/Repressor construct to show presence of GFP/Expressor in gel

### Week 4

Interlab: Scheduled date to use plate reader to measure fluorescence.

Made 16 LB Agar plates with chloramphenicol

# AUGUST

### Week 1

Interlab: Made fluorescent stock; followed the Interlab protocol to prepare and plate samples and controls to be
incubated.

### Week 2

Interlab: all plates except for the negative (as expected), TD 2, and TD 3 grew colonies. Made 16 more LB agar plates
with chloramphenicol for replating of TD 2 and TD 3. Replated TD 2 and TD 3; no growth on either plate

### Week 3

Interlab: resuspended DNA from Kit 7 for TD 2 and TD 3. Replated TD 2 and TD 3 with new DNA; transformations were
successful.

Autoclaved LB broth in preparation for picking colonies

Counted colonies for control transformation: 4 colonies grew on the positive control (20 microliter) plate

Need the DNA concentration to finish calculating competent cell efficiency

### Week 4

5 New modifications with secondary structure- With these modifications we would have expressors that have cDNA of
lengths

15 base 3prime truncation, 20 base 3prime truncation, 25 base 3prime truncation, 9 base truncation from BOTH ends,
5 base remaining from 3prime – rehashing of Expressor Naught

# SEPTEMBER

### Week 1

Interlab: Measured fluorescence using plate reader and finalized protocol for plate reader. (Fluorescein standard
curve) calibration and standard curve mode (serial dilutions)

### Week 2

Analyzed plate reader data to find that our repressor provided imperfect and incomplete repression

### Week 3

Lab meeting with PI to determine the goals for the team and Wiki

### Week 4

Helped Lambert High School team with the plate reader

Interlab: filled out the three protocol forms for the plate reader and the Excel data sheets

# OCTOBER

### Week 1

Write protocol to begin construct tests with AFB1

### Week 2

Overnight incubation with culture, bacteria, and different concentrations of toxin

Constructs tested: Ea, E3, E3R, and a blank no bacteria with toxin

### Week 3

Adapted previous protocol by applying toxin to media during exponential growth phase in order to stimulate aflatoxin-RNA
binding over the competing RNA-RNA binding

Possible signal but further testing is needed for two different conditions (overnight vs. hour aflatoxin introduction)

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# Team:UGA-Georgia/Experiments

* [![notebook](https://static.igem.org/mediawiki/2017/b/b2/UGA-peanut.svg)](https://2017.igem.org/Team%3AUGA-Georgia)
* ![notebook](https://static.igem.org/mediawiki/2017/6/68/UGA-team.svg)

  Team

  [Team](https://2017.igem.org/Team%3AUGA-Georgia/Team)
  [Collaborators](https://2017.igem.org/Team%3AUGA-Georgia/Collaborators)
  [Attributions](https://2017.igem.org/Team%3AUGA-Georgia/Attributions)
* ![beaker](https://static.igem.org/mediawiki/2017/5/5c/UGA-beaker.svg)

  Project

  [Description](https://2017.igem.org/Team%3AUGA-Georgia#description)
  [Design](https://2017.igem.org/Team%3AUGA-Georgia/Design)

  [InterLab](https://2017.igem.org/Team%3AUGA-Georgia/InterLab)
  [Results](https://2017.igem.org/Team%3AUGA-Georgia/Results)
* [![notebook](https://static.igem.org/mediawiki/2017/d/df/UGA-notebook.svg)

  Notebook](https://2017.igem.org/Team%3AUGA-Georgia/Notebook)
* [![notebook](https://static.igem.org/mediawiki/2017/4/46/UGA-parts.svg)

  Parts](https://2017.igem.org/Team%3AUGA-Georgia/Parts)
* [![notebook](https://static.igem.org/mediawiki/2017/5/55/UGA-safety.svg)

  Safety](https://2017.igem.org/Team%3AUGA-Georgia/Safety)
* ![notebook](https://static.igem.org/mediawiki/2017/8/85/UGA-human-practices.svg)

  Human Practices

  [Public Engagement](https://2017.igem.org/Team%3AUGA-Georgia/Public_Engagement)
  [Survey](https://2017.igem.org/Team%3AUGA-Georgia/Survey)
* ![notebook](https://static.igem.org/mediawiki/2017/e/e3/UGA-achievements.svg)

  Achievements

  [Bronze](https://2017.igem.org/Team%3AUGA-Georgia/Bronze)
  [Silver](https://2017.igem.org/Team%3AUGA-Georgia/Silver)

* [Judging Form](https://igem.org/2017_Judging_Form?team=UGA-Georgia)
* [Wiki Design by: Logan Jahnke](http://loganjahnke.com)
* [University of Georgia iGem Team 2017](https://igem.org/2017_Judging_Form?team=UGA-Georgia)

# Experiment

For this experiment, we were given six test devices, a positive control, and a negative control in our Kit Plates
6 and 7. All of the devices coded for GFP except for the negative control which was used to calculate the baseline
for background noise. Each DNA sample was resuspended and transformed into Escherichia coli DH5α competent cells
using heat shock. After a 1 hour incubation at 200 rpm, the cells were spread onto petri plates containing LB
agar and Chloramphenicol to be incubated again at 37°C overnight. The next morning, the colonies on the 100μl
positive control plate were counted to calculate our competent cell efficiency and verified that our competent
cells had an efficiency that met the iGEM standards (between 1.5x10^8 and 6x10^8 cfu/µg). Single colonies were
picked from each transformation for PCR to verify the part sizes and make glycerol stock.

Immediately following the overnight incubation of the competent cells with the transformed plasmids, 2 colonies were
picked from each plate and inoculated in 5 mL of LB medium and 5µL of Chloramphenicol to grow overnight at 37°C
and 220 rpm. The following day, the cultures were all diluted to a target OD600 of 0.02 in 12 mL of LB medium
and Chloramphenicol in 50 mL falcon tubes. The cultures were further incubated at the same conditions as before
with 500µL samples taken at 0, 2, 4, and 6 hour timepoints. The samples were then measured for OD and FI values
in the plate reader. These values were submitted to iGEM using the Google Form links provided on the iGEM website.

NOTE: Prior to utilizing the plate reader, LUDOX-S40 was used to create a single point reference to obtain a ratiometric
conversion factor to transform the absorbance data into a standard OD600 measurement. The plate reader was then
calibrated using a fluorescein fluorescence standard curve using a dilution series of fluorescein. All calibration
steps followed the Interlab 2017 Plate Reader Protocol, officially set by iGEM.

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# Team:UGA-Georgia/Results

* [![notebook](https://static.igem.org/mediawiki/2017/b/b2/UGA-peanut.svg)](https://2017.igem.org/Team%3AUGA-Georgia)
* ![notebook](https://static.igem.org/mediawiki/2017/6/68/UGA-team.svg)

  Team

  [Team](https://2017.igem.org/Team%3AUGA-Georgia/Team)
  [Collaborators](https://2017.igem.org/Team%3AUGA-Georgia/Collaborators)
  [Attributions](https://2017.igem.org/Team%3AUGA-Georgia/Attributions)
* ![beaker](https://static.igem.org/mediawiki/2017/5/5c/UGA-beaker.svg)

  Project

  [Description](https://2017.igem.org/Team%3AUGA-Georgia#description)
  [Design](https://2017.igem.org/Team%3AUGA-Georgia/Design)

  [InterLab](https://2017.igem.org/Team%3AUGA-Georgia/InterLab)
  [Results](https://2017.igem.org/Team%3AUGA-Georgia/Results)
* [![notebook](https://static.igem.org/mediawiki/2017/d/df/UGA-notebook.svg)

  Notebook](https://2017.igem.org/Team%3AUGA-Georgia/Notebook)
* [![notebook](https://static.igem.org/mediawiki/2017/4/46/UGA-parts.svg)

  Parts](https://2017.igem.org/Team%3AUGA-Georgia/Parts)
* [![notebook](https://static.igem.org/mediawiki/2017/5/55/UGA-safety.svg)

  Safety](https://2017.igem.org/Team%3AUGA-Georgia/Safety)
* ![notebook](https://static.igem.org/mediawiki/2017/8/85/UGA-human-practices.svg)

  Human Practices

  [Public Engagement](https://2017.igem.org/Team%3AUGA-Georgia/Public_Engagement)
  [Survey](https://2017.igem.org/Team%3AUGA-Georgia/Survey)
* ![notebook](https://static.igem.org/mediawiki/2017/e/e3/UGA-achievements.svg)

  Achievements

  [Bronze](https://2017.igem.org/Team%3AUGA-Georgia/Bronze)
  [Silver](https://2017.igem.org/Team%3AUGA-Georgia/Silver)

* [Judging Form](https://igem.org/2017_Judging_Form?team=UGA-Georgia)
* [Wiki Design by: Logan Jahnke](http://loganjahnke.com)
* [University of Georgia iGem Team 2017](https://igem.org/2017_Judging_Form?team=UGA-Georgia)

# Results

![results3](https://static.igem.org/mediawiki/2017/7/7d/E3results1.jpeg)
![results3](https://static.igem.org/mediawiki/2017/9/9f/E3rresults2.jpeg)
![results3](https://static.igem.org/mediawiki/2017/f/fd/Rresults3.jpeg)
![results3](https://static.igem.org/mediawiki/2017/6/63/Earesults4.jpeg)

### Conclusion

From our data, it is clear that the final biosensor (E3R) has fluorescence without the prescence of toxin, AFB1. This background fluorescence, stemming from imperfect repression, was low enough when compared to the max theoretical fluorescence (E3) that there existed a wide enough range to see a response to AFB1 if this response exists. Though there existed a wide enough range to observe a response, no response was observed. This was the case even when particularly high levels of the toxin were utilized (up to 10 ppm). This could suggest that the affinity of the repressor for AFB1 is prohibitively low as compared to the affinity of the repressor for the expressor. It also might suggest that the cytoplasmic conditions are inhibitory when it comes to the interaction between the repressor and AFB1.

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