iGEM 2020 — Team Lambert_GA

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lambert GA/Notebook

[![](https://static.igem.org/mediawiki/2020/c/c6/T--Lambert_GA--logowithname.png)](https://2020.igem.org/Team%3ALambert_GA)

* TEAM

  [TEAM MEMBERS](https://2020.igem.org/Team%3ALambert_GA/Team)
  [ATTRIBUTIONS](https://2020.igem.org/Team%3ALambert_GA/Attributions)
* PROJECT

  [DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Description)
  [DESIGN](https://2020.igem.org/Team%3ALambert_GA/Design)
  [CONTRIBUTION](https://2020.igem.org/Team%3ALambert_GA/Contribution)
  [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3ALambert_GA/Implementation)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ALambert_GA/Engineering)
  [PROOF OF CONCEPT](https://2020.igem.org/Team%3ALambert_GA/Proof_Of_Concept)
  [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3ALambert_GA/Excellence)
* WETLAB

  [PARTS LIBRARY](https://2020.igem.org/Team%3ALambert_GA/PartsLibrary)
  [PARTS DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Parts)
  [NUTRIENT MIX](https://2020.igem.org/Team%3ALambert_GA/NutrientMix)
  [PHO](https://2020.igem.org/Team%3ALambert_GA/Pho)
  [NAR](https://2020.igem.org/Team%3ALambert_GA/Nar)
  [IRON](https://2020.igem.org/Team%3ALambert_GA/Iron)
  [EXPERIMENTS](https://2020.igem.org/Team%3ALambert_GA/Experiments)
  [NOTEBOOK](https://2020.igem.org/Team%3ALambert_GA/Notebook)
  [RESULTS](https://2020.igem.org/Team%3ALambert_GA/Results)
* DRYLAB

  [HARDWARE](https://2020.igem.org/Team%3ALambert_GA/Hardware)
  [SOFTWARE](https://2020.igem.org/Team%3ALambert_GA/Software)
  [MEASUREMENT](https://2020.igem.org/Team%3ALambert_GA/Measurement)
  [MODEL](https://2020.igem.org/Team%3ALambert_GA/Model)
* HUMAN PRACTICES

  [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ALambert_GA/Human_Practices)
  [SCIENCE COMMUNICATION](https://2020.igem.org/Team%3ALambert_GA/Education)
  [COLLABORATIONS](https://2020.igem.org/Team%3ALambert_GA/Collaborations)
  [SAFETY](https://2020.igem.org/Team%3ALambert_GA/Safety)
  [SUSTAINABLE DEVELOPMENT](https://2020.igem.org/Team%3ALambert_GA/Sustainable)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?team=Lambert_GA)
* [POSTER](https://2020.igem.org/Team%3ALambert_GA/Poster)

![](https://static.igem.org/mediawiki/2020/e/ef/T--Lambert_GA--NotebookCover.jpg)

NOTEBOOK

[

](https://static.igem.org/mediawiki/2020/d/d0/T--Lambert_GA--scroll.mp4)
[NUTRIENT MIX NOTEBOOK](#nutrient)
[Pho NOTEBOOK](#pho)
[Nar NOTEBOOK](#nar)

# NUTRIENT MIX NOTEBOOK

# Pho NOTEBOOK

# Nar NOTEBOOK

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lambert GA/Engineering

[![](https://static.igem.org/mediawiki/2020/c/c6/T--Lambert_GA--logowithname.png)](https://2020.igem.org/Team%3ALambert_GA)

* TEAM

  [TEAM MEMBERS](https://2020.igem.org/Team%3ALambert_GA/Team)
  [ATTRIBUTIONS](https://2020.igem.org/Team%3ALambert_GA/Attributions)
* PROJECT

  [DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Description)
  [DESIGN](https://2020.igem.org/Team%3ALambert_GA/Design)
  [CONTRIBUTION](https://2020.igem.org/Team%3ALambert_GA/Contribution)
  [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3ALambert_GA/Implementation)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ALambert_GA/Engineering)
  [PROOF OF CONCEPT](https://2020.igem.org/Team%3ALambert_GA/Proof_Of_Concept)
  [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3ALambert_GA/Excellence)
* WETLAB

  [PARTS LIBRARY](https://2020.igem.org/Team%3ALambert_GA/PartsLibrary)
  [PARTS DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Parts)
  [NUTRIENT MIX](https://2020.igem.org/Team%3ALambert_GA/NutrientMix)
  [PHO](https://2020.igem.org/Team%3ALambert_GA/Pho)
  [NAR](https://2020.igem.org/Team%3ALambert_GA/Nar)
  [IRON](https://2020.igem.org/Team%3ALambert_GA/Iron)
  [EXPERIMENTS](https://2020.igem.org/Team%3ALambert_GA/Experiments)
  [NOTEBOOK](https://2020.igem.org/Team%3ALambert_GA/Notebook)
  [RESULTS](https://2020.igem.org/Team%3ALambert_GA/Results)
* DRYLAB

  [HARDWARE](https://2020.igem.org/Team%3ALambert_GA/Hardware)
  [SOFTWARE](https://2020.igem.org/Team%3ALambert_GA/Software)
  [MEASUREMENT](https://2020.igem.org/Team%3ALambert_GA/Measurement)
  [MODEL](https://2020.igem.org/Team%3ALambert_GA/Model)
* HUMAN PRACTICES

  [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ALambert_GA/Human_Practices)
  [SCIENCE COMMUNICATION](https://2020.igem.org/Team%3ALambert_GA/Education)
  [COLLABORATIONS](https://2020.igem.org/Team%3ALambert_GA/Collaborations)
  [SAFETY](https://2020.igem.org/Team%3ALambert_GA/Safety)
  [SUSTAINABLE DEVELOPMENT](https://2020.igem.org/Team%3ALambert_GA/Sustainable)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?team=Lambert_GA)
* [POSTER](https://2020.igem.org/Team%3ALambert_GA/Poster)

![](https://static.igem.org/mediawiki/2020/5/58/T--Lambert_GA--EngineeringSuccessCover.jpeg)

ENGINEERING SUCCESS

[

](https://static.igem.org/mediawiki/2020/d/d0/T--Lambert_GA--scroll.mp4)
[NAR ENGINEERING SUCCESS](#nar)

# NAR ENGINEERING SUCCESS

![](https://static.igem.org/mediawiki/2020/b/b9/T--Lambert_GA--NARKey.png)

*Figure 1. Diagram of the Nar Engineering Success Workflow*

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lambert GA/Experiments

[![](https://static.igem.org/mediawiki/2020/c/c6/T--Lambert_GA--logowithname.png)](https://2020.igem.org/Team%3ALambert_GA)

* TEAM

  [TEAM MEMBERS](https://2020.igem.org/Team%3ALambert_GA/Team)
  [ATTRIBUTIONS](https://2020.igem.org/Team%3ALambert_GA/Attributions)
* PROJECT

  [DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Description)
  [DESIGN](https://2020.igem.org/Team%3ALambert_GA/Design)
  [CONTRIBUTION](https://2020.igem.org/Team%3ALambert_GA/Contribution)
  [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3ALambert_GA/Implementation)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ALambert_GA/Engineering)
  [PROOF OF CONCEPT](https://2020.igem.org/Team%3ALambert_GA/Proof_Of_Concept)
  [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3ALambert_GA/Excellence)
* WETLAB

  [PARTS LIBRARY](https://2020.igem.org/Team%3ALambert_GA/PartsLibrary)
  [PARTS DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Parts)
  [NUTRIENT MIX](https://2020.igem.org/Team%3ALambert_GA/NutrientMix)
  [PHO](https://2020.igem.org/Team%3ALambert_GA/Pho)
  [NAR](https://2020.igem.org/Team%3ALambert_GA/Nar)
  [IRON](https://2020.igem.org/Team%3ALambert_GA/Iron)
  [EXPERIMENTS](https://2020.igem.org/Team%3ALambert_GA/Experiments)
  [NOTEBOOK](https://2020.igem.org/Team%3ALambert_GA/Notebook)
  [RESULTS](https://2020.igem.org/Team%3ALambert_GA/Results)
* DRYLAB

  [HARDWARE](https://2020.igem.org/Team%3ALambert_GA/Hardware)
  [SOFTWARE](https://2020.igem.org/Team%3ALambert_GA/Software)
  [MEASUREMENT](https://2020.igem.org/Team%3ALambert_GA/Measurement)
  [MODEL](https://2020.igem.org/Team%3ALambert_GA/Model)
* HUMAN PRACTICES

  [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ALambert_GA/Human_Practices)
  [SCIENCE COMMUNICATION](https://2020.igem.org/Team%3ALambert_GA/Education)
  [COLLABORATIONS](https://2020.igem.org/Team%3ALambert_GA/Collaborations)
  [SAFETY](https://2020.igem.org/Team%3ALambert_GA/Safety)
  [SUSTAINABLE DEVELOPMENT](https://2020.igem.org/Team%3ALambert_GA/Sustainable)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?team=Lambert_GA)
* [POSTER](https://2020.igem.org/Team%3ALambert_GA/Poster)

![](https://static.igem.org/mediawiki/2020/4/47/T--Lambert_GA--ExperimentsPageHeader.jpg)

EXPERIMENTS

[

](https://static.igem.org/mediawiki/2020/d/d0/T--Lambert_GA--scroll.mp4)

[CLONING PROTOCOL](#cloning)
[NUTRIENT MIX PROTOCOL](#nutrient)

# CLONING PROTOCOL

## **RESTRICTION DIGEST**

| Reagent | Amount |
| --- | --- |
| Restriction Enzyme 1 | 1 uL |
| Restriction Enzyme 2 | 1 uL |
| 10x Cutsmart Buffer | 5 uL |
| Insert or Vector DNA | 1 ug |
| Nuclease Free Water/ Sterile Water | To 50 uL |

1. Shave ice in styrofoam container
2. Label PCR tubes
3. Add all reagents to tube
4. Incubate for 30 minutes at 37°C
5. Incubate for 20 minutes at 65°C

## **LIGATION**

| Reagent | Amount |
| --- | --- |
| T4 Ligase Buffer | 2 uL |
| Vector DNA | 50 ng |
| Insert DNA | 150 ng |
| Nuclease Free Water | To 20 ng |
| T4 DNA Ligase | To 50 uL |

1. Shave ice
2. Label all PCR tubes
3. Add all of the reagents to your tube
4. Incubate for 10 min at room temperature
5. Incubate for 10 min at 65°C

## **TRANSFORMATION**

1. Label tubes with initials, date, and what is being transformed
2. Place all tubes on ice
3. Put SOC/LB and antibiotic plates in the incubator to warm at 37°C
4. Pipette 1ul of the desired plasmid(s) and 1ul of PUC19 into the corresponding labeled transformation tubes
5. Thaw competent cells on ice for 10 minutes or just until the cells inside are moving when you flick the tube
6. Pipette 50ul of competent cells into each labeled transformation tube on ice
7. Incubate tubes on ice for 30 minutes
8. Heat shock tubes at 42°C for 45 seconds
9. Put on ice 2 minutes
10. Add 950ul of SOC/LB media into each tube with cells
11. Place tubes shaking in the incubator for 60 minutes at 37°C
12. Pipette 120-150ul of cells with SOC/LB media onto warm plates with the correct antibiotics
13. Place antibiotic plates in the incubator at 37°C for 24 hours to let cells grow

## **INOCULATION FOR CULTURES**

1. Prepare Liquid LB
2. When ready to grow culture, add Liquid LB to a tube or flask and add your antibiotic that your cells are resistant to the correct concentration to create a 1 milliliter of LB to 1 microliter of antibiotic (typically use 5 ml: 5 ul)
3. Using a sterile pipette tip select a single colony from your LB agar plate
4. Drop the tip into liquid LB and antibiotic, then swirl
5. Cover culture with sterile aluminum foil or cap that is not airtight
6. Incubate culture at 37℃ for 12-18 hours in a shaking incubator
7. After incubation, check for growth
8. For long term, storage continue with creating a glycerol stock

## **MINIPREP**

1. Grow 1-5 mL culture overnight in a 10mL-20mL culture tube.
2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
3. Add 250 uL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
4. Add 250 uL of solution 2. Invert until there is clear lysate.
5. Add 350 uL to solution 3, insert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
6. Insert mini column into a 2 mL collection tube.
7. Transfer the clear supernatant into a mini-column using a micropipette. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
8. Add 500 uL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
9. Add 700 uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 30 seconds, Discard filtrate, and reuse collection tube.
10. Centrifuge empty mini-column at 13,0000xg for 2 minutes to remove ethanol.
11. Transfer mini-column to a nuclease-free 1.5 mL microcentrifuge tube.
12. Add 50 uL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000xg for 60 seconds.
13. Store eluted DNA at -20℃

## **GEL ELECTROPHORESIS**

### **MAKING GEL:**

1. Mix 0.5g of agarose with 50ml of 1x TAE buffer in a 100-250ml Erlenmeyer flask
2. Heat up the solution until it turns clear
3. Let the solution cool down until it reaches 65°C
4. Once the solution measures 65°C, add 6ul of SybrSafe in and gently shake the flask to mix the solution
5. Pour the solution into a gel chamber and put in the gel comb
6. Wait for the gel to solidify before loading

### **RUNNING THE GEL:**

7. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red
8. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel
9. On a piece of parafilm, mix 5ul of DNA and 1ul of purple loading dye using a micropipette
10. Add the mixed 6ul solution into each well
11. Add 6ul of 2-log ladder into an empty well
12. Connect the electrodes by closing the gel chamber and connecting the to the power supply
13. Set power supply for 90 volts and 45 minutes
14. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber
15. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder

## **PCR**

| Reagent | Amount |
| --- | --- |
| Sterile water | To 25 uL (normally 9uL) |
| Q5 High Fidelity Master Mix | 12.5 uL |
| 10 uM diluted forward primer | 1.25 uL |
| 10 uM diluted reverse primer | 1.25 uL |
| Template DNA | 1 uL |

1. Add reagents to labeled PCR tube
2. Set thermocycler with new annealing temperature and elongation times (annealing time and temperature depends on your part to PCR)
3. Run PCR

## **GIBSON ASSEMBLY**

### **PART 1: ASSEMBLY**

| Reagent | Amount |
| --- | --- |
| Sterile water | To 20 uL |
| Gibson assembly master mix (2x) | 10 uL |
| PCR fragments | Variable (dep on calculation done above - norm 1 uL) |
| Linearized Vectors | Variable (dep on calculation done above - norm 1 uL) |

1. Add all reagents into labeled PCR tubes
2. Add 10 uL Gibson positive control and 10 uL master mix to another labeled PCR tube for your control
3. Incubate in thermocycler at 50 degrees C for 15 mins when assembling 2 or 3 fragments or 50 degrees C for 60 mins when assembling 4-6 fragments
4. Store at -20 degrees C

### **PART 2: TRANSFORMATION**

- Add 2 uL of Gibson/ligated DNA to microcentrifuge tubes and 1 uL Puc into another tube
- Add 50 uL competent cells to microcentrifuge tubes and mix by pipetting up and down 4-5 times
- Place microcentrifuge tubes on ice for 30 minutes
- Heat shock at 42 degrees for 30 seconds
- Transfer tubes on ice for 2 minutes
- Add 950 uL SOC media to tubes
- Place tubes in shaking incubator at 37 degrees C for 60 mins
- Plate 200 uL cells onto antibiotic plates (Gibson Positive Control goes on a carb plate)
- Incubate plates overnight at 37 degrees C

## **COLONY PCR**

| Reagent | Amount |
| --- | --- |
| Q5 High-Fidelity 2X Master Mix | 12.5uL |
| 10uM Forward Primer VF2 | 1.25uL |
| 10uM Reverse Primer VR | 1.25uL |
| Template DNA | 1.25 uL |
| Nuclease-Free Water | To 25uL |

1. Choose colony and dilute with 40uL of water
2. Take 1uL out for the colony PCR
3. Put all together in PCR tube
4. Place in Thermocycler

## **PCR CLEANUP**

1. Add 1:1 volume of binding buffer to completed PCR mixture
2. Transfer up to 800 uL to GeneJET purification column
3. Centrifuge for 60 seconds and discard flow through
4. Add 700 uL wash buffer to the purification column
5. Centrifuge for 60 seconds and discard flow through
6. Centrifuge again for 60 seconds (dry spin) and discard flow through
7. Transfer purification column to 1.5 uL microcentrifuge tube
8. Add 30 uL elution buffer
9. Wait 5 minutes
10. Centrifuge for 1 minutes
11. Store at -20 degrees C

# NUTRIENT MIX PROTOCOL

## **PROTOCOL (FOR BOTH NITRITE, NITRATE) IN TECHNICAL TRIPLE KITS**

1. Autoclave 250ml flask (12)

1. 6 for different concentrations of nitrite (0ppm,60ppm, 120ppm, 180ppm, 240ppm, 300ppm)
2. 6 for different concentrations for nitrate (0ppm,60ppm, 120ppm, 180ppm, 240ppm, 300ppm)

2. Add 100ul of pre-grown cells into 1 liquid culture tube

1. Add 100ml of plain lb into a 250 flask
2. Add 5ml of the cell stock

1. Add 60ml of diH2O with a cell colony

3. Grow the flask for 24 hours in the shaking incubator

3. Add 100ml of LB into 6 unlabeled flasks and add 10ul from liquid culture tube
4. Grow flasks for 12 hours in shaking incubator
5. After 12 hours, transfer 50ml to each of the sterile, unused flasks
6. Label each flask and 37 cuvettes \*36 for the triplicates\*

1. Take 1ml from each flask and add it to the corresponding cuvette
2. 36 for cells and 1 for plain lb as a negative control (blank)

7. Measure OD600 in triplicates

1. Take measurements 3 times for each cuvette then average data

8. After measurement, add X grams of nitrite and nitrate to each flask
9. Grow flasks for 4 hours in shaking incubator
10. Take measurement again (repeat step 7)
11. Grow flasks for 12-16 hours in shaking incubator
12. Repeat step 7
13. Grow flasks for 12 hours in shaking incubator
14. Repeat step 7

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lambert GA/Results

[![](https://static.igem.org/mediawiki/2020/c/c6/T--Lambert_GA--logowithname.png)](https://2020.igem.org/Team%3ALambert_GA)

* TEAM

  [TEAM MEMBERS](https://2020.igem.org/Team%3ALambert_GA/Team)
  [ATTRIBUTIONS](https://2020.igem.org/Team%3ALambert_GA/Attributions)
* PROJECT

  [DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Description)
  [DESIGN](https://2020.igem.org/Team%3ALambert_GA/Design)
  [CONTRIBUTION](https://2020.igem.org/Team%3ALambert_GA/Contribution)
  [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3ALambert_GA/Implementation)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ALambert_GA/Engineering)
  [PROOF OF CONCEPT](https://2020.igem.org/Team%3ALambert_GA/Proof_Of_Concept)
  [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3ALambert_GA/Excellence)
* WETLAB

  [PARTS LIBRARY](https://2020.igem.org/Team%3ALambert_GA/PartsLibrary)
  [PARTS DESCRIPTION](https://2020.igem.org/Team%3ALambert_GA/Parts)
  [NUTRIENT MIX](https://2020.igem.org/Team%3ALambert_GA/NutrientMix)
  [PHO](https://2020.igem.org/Team%3ALambert_GA/Pho)
  [NAR](https://2020.igem.org/Team%3ALambert_GA/Nar)
  [IRON](https://2020.igem.org/Team%3ALambert_GA/Iron)
  [EXPERIMENTS](https://2020.igem.org/Team%3ALambert_GA/Experiments)
  [NOTEBOOK](https://2020.igem.org/Team%3ALambert_GA/Notebook)
  [RESULTS](https://2020.igem.org/Team%3ALambert_GA/Results)
* DRYLAB

  [HARDWARE](https://2020.igem.org/Team%3ALambert_GA/Hardware)
  [SOFTWARE](https://2020.igem.org/Team%3ALambert_GA/Software)
  [MEASUREMENT](https://2020.igem.org/Team%3ALambert_GA/Measurement)
  [MODEL](https://2020.igem.org/Team%3ALambert_GA/Model)
* HUMAN PRACTICES

  [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ALambert_GA/Human_Practices)
  [SCIENCE COMMUNICATION](https://2020.igem.org/Team%3ALambert_GA/Education)
  [COLLABORATIONS](https://2020.igem.org/Team%3ALambert_GA/Collaborations)
  [SAFETY](https://2020.igem.org/Team%3ALambert_GA/Safety)
  [SUSTAINABLE DEVELOPMENT](https://2020.igem.org/Team%3ALambert_GA/Sustainable)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?team=Lambert_GA)
* [POSTER](https://2020.igem.org/Team%3ALambert_GA/Poster)

![](https://static.igem.org/mediawiki/2020/f/f5/T--Lambert_GA--resultsheader.jpg)

RESULTS

[

](https://static.igem.org/mediawiki/2020/d/d0/T--Lambert_GA--scroll.mp4)
[CHARACTERIZATION](#characterization)
[REFERENCES](#references)

# CHARACTERIZATION

## **PURPOSE OF CHARACTERIZATION**

Based on Lambert iGEM’s ODE model, the 4uM threshold of BBa\_K2447000 seems to be less strict than described above. Extracellular phosphate concentrations greater than 4uM, even in the range of 100uM, will still result in minimal GFP expression. To improve upon the existing characterization of the phosphate sensor, Lambert iGEM tested with a greater number of phosphate concentrations from 0 to 100uM in intervals of 20uM.

## **EXPERIMENTAL DESIGN**

In order to improve part BBa\_K116404 created by NYMU-Taipei 2008, NUS Singapore iGEM 2017 created part BBa\_K2447000 by replacing the weaker RBS BBa\_B0032 with a stronger RBS BBa\_B0034. This allowed the BBa\_K2447000 extracellular phosphate sensor to be much more sensitive to various phosphate concentrations from 0uM to 1000uM.

Through research, the team discovered that typical phosphate levels in aquaponics systems range from 10ppm to 40ppm [3]. Using dimensional analysis, the team concluded that maintaining a phosphate concentration between 50uM and 100uM is ideal for plants and fish to coexist. To add detailed characterization of part BBa\_K2447000 on phosphate concentrations specifically targeted for use in aquaponics systems, Lambert iGEM tested GFP expression of the phosphate sensor on extracellular phosphate levels from 0uM to 100uM in intervals of 20uM.

## **EXPERIMENTAL PROCEDURE**

The characterization protocol began with the team’s biosensor cells being grown in chloramphenicol LB for 24 hours and later diluted to an OD600 value of 0.4. Then, the cells were pelleted and resuspended into MOPS media, which has minimal phosphate concentration relative to LB. To the 5 mL resuspension, the team added different phosphate concentrations between 0 to 100uM and waited 3 hours for GFP to be expressed. In order to measure the GFP expression, Lambert iGEM used a plate reader from Styczynski Research Group at Georgia Institute of Technology.

## **CHARACTERIZATION CURVE**

![](https://static.igem.org/mediawiki/2020/3/3f/T--Lambert_GA--pho_characterization_curve.png)

*Figure 1. Characterization curve showing the relationship between phosphate concentrations between 0 to 100uM and fluorescence/OD600 measured by a plate reader.*

![](https://static.igem.org/mediawiki/2020/a/a8/T--Lambert_GA--pho_model_short.png)

*Figure 2. Prediction of relationship between GFP expression and phosphate concentrations ranging from 0 to 100uM made by deterministic ODE model.*

Using data from the plate reader, Lambert iGEM created a characterization curve showing the relationship between phosphate concentrations and fluorescence/OD600. For phosphate concentrations ranging from 0 to 80uM, the decreasing trend in fluorescence/OD600 closely resembled Lambert iGEM’s ODE model prediction, shown in Figure 2. The fluorescence value for the 100uM phosphate concentration did not match the prediction from the model because the phosphate media was diluted improperly, causing its measured GFP expression to be higher than expected. Due to time constraints in the lab, the team was not able to conduct further testing and decided to use the characterization data for only 0 to 80uM of phosphate.

## **SAMPLE TESTING**

Hypothetically, using the biosensor cells, aquaponics farmers should be able to identify the phosphate concentrations of unknown samples by utilizing the characterization curve. For instance, if the biosensor cells expressed Y arbitrary units of GFP, the user could find a point (X, Y) in the characterization curve in which X would be the corresponding phosphate concentration. This method would be applicable in identifying the phosphate concentration in unknown samples from real aquaponics systems such as a nutrient fertilizer mix, waste water from fish, and even tap water. Lambert iGEM did not have enough time to perform sample testing, but will be conducting experiments next year.

# REFERENCES

[1] Storey, N. (2017, December 13). The Most Important Things To Know About Phosphorus. Retrieved October 03, 2020, from https://university.upstartfarmers.com/blog/most-important-things-about-phosphorus.