iGEM 2019 — Team UTArlingtonTexasUSA

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# Team:UTArlingtonTexasUSA/Notebook

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PROJECT

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[Design](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Design)
[Experiments](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Experiments)
[Notebook](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Notebook)
[Results](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Results)
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PARTS

[Parts Overview](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Parts)

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

HUMAN PRACTICES

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

AWARDS

[Hardware](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Hardware)
[Model](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Model)

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

# NOTEBOOK

### January

Attend Engineering Student Council meetings to recruit members and speak about SynBio and iGEM.

### February

Held meetings on our campus with the Genetic Engineering Society. Invited students of all majors to inform them of the competition. Additionally, we help brainstorm sessions between divided teams to think of projects we could potentially conduct during the fall.

### March

Vote on team structure. Developed two teams; one team which would work on the physical hardware aspect; the second team would focus on designing and executing lab testing for the project. Run feasibility project checks with team advisors to get prospective on projects with highest potential for success.

### April

Complete fundraising for team registration. Begin registering ideas for potential products. Finally, we ensured that all members who would be participating in lab work completed all safety certifications for BSL-2 conditions.

### May

Begin lab work and training for those who needed it, protocol assignments, summer lab schedule, and work requirements are established. Begin compiling information for wiki.

### June

Re-visit the drawing board to refine our project idea and continue to develop microbial fuel cell design using assembly and SolidWorks software. In addition, continue research on different pollutants and biosensing parts that could be applicable for our specific design.

### July

Improve the design of the microbial fuel cell, begin the revised genetic engineering portion, order parts and supplies. Continue lab training for all members. Begin assembling prototypes for fuel cell. Order glass and other accessory materials required for running the fuel cell. Consult and collaborated project with UT IGEM over ideas, project, and jamboree details.

### August

Summer class finals, supplies re-orders, more lab training to perform more extensive techniques, beginning of school. UT Arlington IGEM banner was created. Received funding from College of Engineering and Bioengineering department for travel. Finalizing design and using an epilog helix laser cutter to cut out acrylic frame of the fuel cell.

### September

Continued with the revised genetic engineering portion of our project, maintained working function of our microbial fuel cell, finalized gene assembly methods and finalized a plan to integrate the two components of our project. Some members of our team also attended UT iGEM's Undergraduate Research Symposium. Finalizing travel itineraries. Started creating IGEM Wiki.

### October

Finalize the two portions of our projects separately, make travel plans, and make our poster and presentation.

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# Team:UTArlingtonTexasUSA/Experiments

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PROJECT

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

PARTS

[Parts Overview](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Parts)

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

HUMAN PRACTICES

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

AWARDS

[Hardware](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Hardware)
[Model](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Model)

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

# EXPERIMENTS

Transformation of E. Coli DNA was a vital part of our project as this would allow the E. Coli to become bioluminescent in the presence of the Xylene pollutant. XYLR, GFP, then PHZ was the sequence order which we wanted the DNA to be ligated in. Each pairing of enzymes required specific buffer solutions to raise the probability of cutting success; NEBuffer 2.1, NEBuffer 3.1, and CutSmart being the aforementioned buffers. To ensure their characterization as vectors or inserts, specific enzymes, including Echo R1-HF, Xba1, Spe1, and Pst1, were used to cut the DNA at specific locations. If the DNA of a single protein was cut improperly, it would compromise our efforts in ligating all of the DNA onto a PUC-19 plasmid within a single run. To avoid any expected errors and conserve our enzymes, the XYLR, GFP, PHZ were all independently ligated and transformed into separate PUC-19 plasmids. Once each protein was correctly ligated and transformed onto separate cells, two of them were to be ligated together as our next goal. Our team chose to ligate the PHZ DNA onto the GFP cells first, this required the GFP cell DNA to be cut as a vector and the PHZ cell DNA to be cut as an insert. After the GFPPHZ cells were properly made, we decided to ligate the XYLR DNA with the GFPPHZ DNA in two different ways. In one method, the GFPPHZ was cut as an insert which would be placed into a cut XYLR vector. The other method involved cutting XYLR as an insert and setting it into a cut GFPPHZ vector. This was done to minimize any errors that could’ve occurred when choosing the cutting buffers, using the cutting enzymes, or due to the cut DNA lengths causing compatibility issues. Our genetic modification of E. Coli cells ending once the XYLR, GFP, and PHZ DNA was successfully cut, ligated, and transformed into them.

### E. coli k-12 Culture (1mL)

1. Pipette 1mL of sterile Lysogeny Broth (20g LB/ 1L DI H2O) into a sterile 1mL tube

2. Add 1μL (if 2mL of Lysogeny Broth used, then 2μL) of Tetracycline (1000x)
3. Vortex for 10 seconds
4. Incubate at 37°C overnight

### E. coli k-12 Plate Culture

1. Streak a Tetracycline covered plate with first line colony (E. coli k-12)
2. Allow to incubate at 37°C overnight

### Miniprep (Plasmid Extraction): Puc19 – E. coli k-12

1. Column without caps need column preparation, add 500 μL of column prep buffer to 2 columns
2. Centrifuge for 1 min at 12000 rpm
3. Dump out liquid and tap dry onto a Kim wipe and set aside
4. Take 1mL tubes and fill with 750 μL culture (Puc19 – E. coli k-12 w/ LB) that has grown overnight
5. Centrifuge for 1 min at 12000 rpm
6. Dump out liquid and tap dry onto a Kim wipe
7. Repeat steps 4-6
8. Suspend cells with 250 μL P1 buffer
9. Scrap cells against rack to resuspend cells
10. Lyse cells with 250 μL P2 buffer
11. Invert tubes slowly 6 times
12. Neutralize solution with 350 μL N3 buffer
13. Invert tubes slowly 6 times
14. Centrifuge for 10 mins at 12000 rpm
15. Transfer 700 μL of liquid to each column, leaving behind debris
16. Centrifuge for 1 min at 12000 rpm
17. Dump out liquid and tap dry onto a Kim wipe
18. Repeat steps 14-17
19. Add 700 μL of PE Buffer
20. Centrifuge for 1 min at 12000 rpm
21. Dump out liquid and tap dry onto a Kim wipe
22. Centrifuge empty tube for 2 mins at 12000 rpm
23. Dump out liquid
24. Label 2 new 1 mL tubes (Format: bacteria strain/ vector)
25. Place columns from column tubes to the new Eppendorf tubes
26. Dump out liquid
27. Put column back in tube
28. Add 50 μL DI H2O to the column (drop in the middle of the membrane)
29. Leave for two minutes
30. Centrifuge for 2 minutes at 1200 rpm
31. Place column back in original tube
32. Tube left over in tube is DNA

### DNA Cutting

1. Get the miniprepped cells from the fridge.
2. Determine what will be cut as a vector and what will be cut as an insert to assemble the Xylr-GFP-PHz sequence.
3) Determine what enzymes and buffer solution will be used to create the DNA vector
• If the XYLR plasmid will be made a vector, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
• If the PHZ plasmid will be made a vector, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
• If the GFP plasmid will be made a vector for XYLR, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
• If the GFP plasmid will be made a vector for PHZ, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
• If the GFP-PHZ plasmid will be made a vector for XYLR, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
• If the XYLR-GFP plasmid will be made a vector for PHZ, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
4) Determine what enzymes and buffer solution will be used to create the DNA insert
• If the XYLR plasmid will be made an insert, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
• If the PHZ plasmid will be made an insert, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
• If the GFP plasmid will be made an insert for XYLR, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
• If the GFP plasmid will be made an insert for PHZ, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
• If the GFP-PHZ plasmid will be made an insert for XYLR, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution
• If the XYLR-GFP plasmid will be made an insert for PHZ, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution
5. Insert 43 microL of miniprepped DNA into a clean eppendorf tube. If there is less than 43 microL of DNA, fill to 43 microL with sterile H2O.
6. Defrost, previously determined, buffer solution
7. Insert 5 micro L of defrosted buffer solution to 43 micro L Eppendorf tube
8. Insert 1 micro L of each previously determined enzyme to the, now 48 micro L, Eppendorf tube
· Make sure to keep the enzymes at -20 degrees Celsius at all times, as they can become permanently denatured otherwise
Mix the 50 micro L enzyme, buffer, and DNA mixture well before incubating it for 45 min at 37 degrees Celsius.
· The Eppendorf tube should be incubated fixed in place, preventing over agitation of the DNA
9. Remove the Eppendorf tube from the incubator and label it as “CUT”
10. Store the labelled Eppendorf tube at -20 degrees Celsius for further use

### 0.1% Agarose Gel for Electrophoresis

1. Add 0.3g of Agarose to 30mL 0.5x Tae Buffer
2. Heat for 1 minute (take off heat every 5 seconds), ensure agarose is fully dissolved
3. Add 6μL of Ethidium Bromide (Caution: highly carcinogenic)
4. Equally distribute into wells with mold and remove all bubbles
5. Allow to cool and remove gel

### Electrophoresis Gel Purification

1. Cut out gel to separate the gene of interest
2. Weigh around 0.250 g of gel into separate Eppendorf tubes
3. Add 3x volume of gel in OG buffer (i.e.: 750mL)
4. Incubate at 50°C for 10 mins with 2-minute breaks (invert during breaks)
5. Take out of incubation and add 248 mL of IPA to reaction tube
6. Invert 6 times
7. Centrifuge using mini-prep columns at 12000 rpm for 1 minute
8. Dump out liquid
9. Add 700 μL of PE buffer and centrifuge again for 1 minute
10. Empty tubes and centrifuge tubes for 2 minutes
11. 30 μL DI H2O directly onto tissue membrane and leave for 1 minute
12. Dump out liquid and centrifuge for 2 minutes
13. Label and freeze in -20°C

### PCR Purification

1. Add 5x PB (the volume of PCR present)
2. Mix well
3. Run through column at 12000 rpm for 1 minute
4. Dump out liquid
5. Run 700 μL of PE buffer through column, centrifuge at 12000 rpm for 1 minute
6. Dump out liquid and centrifuge empty tube at 12000 rpm for 1 minute
7. Place column into Eppendorf tube and add 25 μL of DI H2O
8. Centrifuge 12000 rpm for 2 minutes
9. Collect DNA

### Proton Exchange Membrane

1. Add 1.2 g of Agarose and in dissolve heated DI H2O
2. Add 8.9g of Potassium Chloride
3. Mix well and pour into the salt bridgemold.

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# Team:UTArlingtonTexasUSA/Results

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PROJECT

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

PARTS

[Parts Overview](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Parts)

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

HUMAN PRACTICES

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

AWARDS

[Hardware](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Hardware)
[Model](https://2019.igem.org/Team%3AUTArlingtonTexasUSA/Model)

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

# RESULTS

After the construction of the MFC, we simulated the baseline methodology by utilizing methylene-blue as a mediator in conjunction with ferricyanide as an electron acceptor to ensure that a current could be measured. We obtained a current of 1.5 mA (Figure 1) and this measurement allowed us to confirm that the construction of the MFC was capable of supporting whether the hypothesized gene sequence could be accurately tested and implemented for the detection of xylene. The first portion of the gene are constitutive and are always on, so the bacteria continuously is producing the protein of interest, Xylr. This protein initiates a cascade that activates the pyocyanin promoter and a fluorescent protein m.cherry.

![](https://static.igem.org/mediawiki/2019/6/66/T--UTArlingtonTexasUSA--graphresults.jpg)

##### Figure 1

![](https://static.igem.org/mediawiki/2019/d/d0/T--UTArlingtonTexasUSA--pyocyaninspectra.jpg)

##### Measuring the optical density of the de-protonated Pyocyanin (blue) vs that of Pyocyanin in a aqueous solution (red). This optical density chart can be used as a general standard for pH when running the fuel cells. It can help determine the chemical state of Pyocyanin that is produced from the cells.

![](https://static.igem.org/mediawiki/2019/thumb/2/24/T--UTArlingtonTexasUSA--gel2.jpg/809px-T--UTArlingtonTexasUSA--gel2.jpg)

##### This is the PUC19 vector at around 2500bp as observed alongside the 1kb ladder.

### Future Plans:

* Create fuel cell that can detect multiple BTEX compounds simultaneously.
* Scale up fuel cell design size and amplify sensitivity of biosensing capabilities for increased viability for large-scale applications
* Commercialize microbial fuel cell for public and private use
* Develop a superior cell capable of fluorescence spectrum-calibration to quantify the activation of m.cherry and GFP
* Use Golden Gate Cloning method to ligate gene sequences in more efficient manner