iGEM 2017 — Team UNebraska-Lincoln

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:UNebraska-Lincoln/Notebook

# UNL 2017

## Helping reduce methane emissions from livestock

[![](https://static.igem.org/mediawiki/2017/8/86/T--UNebraska-Lincoln--smallLogo_2017.png)](https://2017.igem.org/Team%3AUNebraska-Lincoln)

[Team](https://2017.igem.org/Team%3AUNebraska-Lincoln/TeamLanding)

[Team](https://2017.igem.org/Team%3AUNebraska-Lincoln/Team)
[Collaborations](https://2017.igem.org/Team%3AUNebraska-Lincoln/Collaborations)

[Project](https://2017.igem.org/Team%3AUNebraska-Lincoln/ProjectLanding)

[Description](https://2017.igem.org/Team%3AUNebraska-Lincoln/Description)
[Design](https://2017.igem.org/Team%3AUNebraska-Lincoln/Design)
[Experiments](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments)
[Notebook](https://2017.igem.org/Team%3AUNebraska-Lincoln/Notebook)
[InterLab](https://2017.igem.org/Team%3AUNebraska-Lincoln/InterLab)
[Contribution](https://2017.igem.org/Team%3AUNebraska-Lincoln/Contribution)
[Model](https://2017.igem.org/Team%3AUNebraska-Lincoln/Model)
[Results](https://2017.igem.org/Team%3AUNebraska-Lincoln/Results)
[Attributions](https://2017.igem.org/Team%3AUNebraska-Lincoln/Attributions)

[Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/PartsLanding)

[Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Parts)
[Basic Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Composite_Part)

[Safety](https://2017.igem.org/Team%3AUNebraska-Lincoln/Safety)

[Human Practices](https://2017.igem.org/Team%3AUNebraska-Lincoln/HumanLanding)

[Silver HP](https://2017.igem.org/Team%3AUNebraska-Lincoln/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3AUNebraska-Lincoln/HP/Gold_Integrated)

![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)

# Notebook

Detailed protocols can be found on our [experiments page.](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments)

## Week 1

Training week 1:
All team members participated in a two week training program. We learned how to create [LB Broth](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments), and [agar](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) and how to create and store [agar plates](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments). Training was done on how to inoculate an LB culture and how to perform chemical and electro transformations. [PCR (Polymerase Chain Reaction)](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) was performed on a mCherry gene for a preliminary lesson on how the process is done.

##### PCR of mCherry:

![](https://static.igem.org/mediawiki/2017/2/2d/T--UNebraska-Lincoln--notebook_1_2017.png)

## Week 2

Training week 2:
The team training was continued this week. We worked on [restriction enzyme digestion](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on the mCherry gene in preparation for molecular cloning of the gene into a different vector. Training was done on setting up agarose gel and using the [electrophoresis](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) equipment to isolate the mCherry gene.

##### Restriction enzyme product:

![](https://static.igem.org/mediawiki/2017/4/4e/T--UNebraska-Lincoln--notebook_2_2017.png)

##### PCR temperature gradient product:

![](https://static.igem.org/mediawiki/2017/9/9c/T--UNebraska-Lincoln--notebook_3_2017.png)

##### Colony PCR product of mCherry gene:

![](https://static.igem.org/mediawiki/2017/c/ce/T--UNebraska-Lincoln--notebook_4_2017.png)

##### Amplification PCR product:

![](https://static.igem.org/mediawiki/2017/e/ed/T--UNebraska-Lincoln--notebook_5_2017.png)

##### PCR amplification for sequencing:

![](https://static.igem.org/mediawiki/2017/b/b2/T--UNebraska-Lincoln--notebook_6_2017.png)

## Week 3

We spent this week waiting for our DNA parts to be shipped. In the meantime we had deliberations and meetings with Dr. Samodha C. Fernando to gather details about the implementation of our project. Our project was researched in more detail. We started to implement Dr. Fernando’s feedback into our project. We also used this week to look more into possible human practice events.

## Week 4

While we were waiting for our parts to be shipped and continuing research we had a meeting with Quantified Ag (cattle based startup) to get more information about how the cattle industry is ran and how our project could fit in. We started our visits to a local museum and set up a booth with information on global warming and our iGEM project. We had a few skype calls with other iGEM teams from Pakistan, Mexico and Boston, to talk about our projects and possible collaborations.

## Week 5

Nic and Tyler prepared the plasmid pSB1C3 by culturing and [miniprep-ing](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) to isolate the plasmid. The plasmid was digested at the XbaI and SpeI sites in preparation for SLIC ligation. [SLIC](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) was performed after the three gene inserts were cut at the same positions. H (Nitrite Reductase nrfAH) and B(Bromoperoxidase) were transformed while nrfA had to go through the SLIC process again.

##### Restriction Digest of pSB1C3:

![](https://static.igem.org/mediawiki/2017/5/5c/T--UNebraska-Lincoln--notebook_7_2017.png)

## Week 6

Tyler and Nic prepared glycerol stock of the inoculated colonies from the “B” and the “H” transformed cultures. Cultures were then created from the stock and the plasmid was purified. The B plasmid was digested at the AatII and HindIII binding locations, whereas the H plasmid was digested at the BpvcI and SalI binding sites. [Gel electrophoresis](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) showed that the size of the cut gene was not at the correct locations. PCR on the gene blocks was tried anyway in preparation for sequencing. The A (Nitrite Reductase nrfA) gene was transformed and a glycerol stock was made after culturing.

##### Restriction Enzyme Digest to test for successful transformation:

![](https://static.igem.org/mediawiki/2017/3/3a/T--UNebraska-Lincoln--notebook_8_2017.png)

## Week 7

Jessica performed [PCR](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on A, B, and H samples so that the team could get a higher concentration for sequencing. The team learned from sequencing that A was possibly correct so we set it aside for further sequencing. B and H were likely incorrect so we went through steps to perform [SLIC](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) again. Nic digested the B and H vectors and Jessica digested the RFP to make an empty vector. After that Nic performed the SLIC procedure and then transformed our newly cloned B and H plasmids while Jessica learned how to do protein purifications and western blots. This week Jessica also performed [colony PCR](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on the newly cloned B colonies. One promising match was found and was further sequenced.

##### PCR amplification of each gene insert: A, B, H:

![](https://static.igem.org/mediawiki/2017/0/0f/T--UNebraska-Lincoln--notebook_9_2017.png)

##### Documentation of which sections were extracted for [DNA recovery](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) and sequence preparation:

![](https://static.igem.org/mediawiki/2017/2/2a/T--UNebraska-Lincoln--notebook_10_2017.png)

##### Justin's training protein purification:

![](https://static.igem.org/mediawiki/2017/0/08/T--UNebraska-Lincoln--notebook_11_2017.png)

##### Restriction digest on vectors B and H using XbaI and HindIII:

![](https://static.igem.org/mediawiki/2017/9/92/T--UNebraska-Lincoln--notebook_12_2017.png)

##### Restriction Digest of pSB1C3-RFP using XbaI and SpeI:

![](https://static.igem.org/mediawiki/2017/2/2a/T--UNebraska-Lincoln--notebook_13_2017.png)

##### Electrophoresis performed on whole vector from glycerol stocks: B1,B2, H1, and H2:

![](https://static.igem.org/mediawiki/2017/c/c5/T--UNebraska-Lincoln--notebook_14_2017.png)

## Week 8

More [restriction enzyme digests](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) and sequencing were done by Nic and Jessica to gather more information to decide if B and H were cloned correctly. While this was being done more empty vector was prepared by Nick Flaxbeard just incase we had to do SLIC again. Sequencing results for A were promising so Jessica performed a [protein purification](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on the A1 colony while continuing to send in sequencing samples for the B8 colony.

##### Restriction digest using...B10:

![](https://static.igem.org/mediawiki/2017/8/8c/T--UNebraska-Lincoln--notebook_15_2017.png)

##### PCR amplification on B8 and on the H insert:

![](https://static.igem.org/mediawiki/2017/5/5a/T--UNebraska-Lincoln--notebook_16_2017.png)

![](https://static.igem.org/mediawiki/2017/8/8f/T--UNebraska-Lincoln--notebook_17_2017.png)

![](https://static.igem.org/mediawiki/2017/e/e8/T--UNebraska-Lincoln--notebook_18_2017.png)

![](https://static.igem.org/mediawiki/2017/8/88/T--UNebraska-Lincoln--notebook_19_2017.png)

##### PCR Amplification of A1, B1, H2:

![](https://static.igem.org/mediawiki/2017/d/d3/T--UNebraska-Lincoln--notebook_20_2017.png)

##### Protein Purification A1:

![](https://static.igem.org/mediawiki/2017/9/98/T--UNebraska-Lincoln--notebook_21_2017.png)

##### Failed H insert PCR amplification:

![](https://static.igem.org/mediawiki/2017/c/c3/T--UNebraska-Lincoln--notebook_22_2017.png)

![](https://static.igem.org/mediawiki/2017/e/e2/T--UNebraska-Lincoln--notebook_23_2017.png)

## Week 9

Jessica performed multiple gradient [PCRs](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on H to test if it could be amplified. These were not working so our team felt safe to assume that H was not cloned properly and Nic began working on recloning H using a different method. While this was being done Nick Flaxbeard started characterizing the A1 colony using the [Nessler's test](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments).

##### Another failed PCR amplification of H:

![](https://static.igem.org/mediawiki/2017/2/2f/T--UNebraska-Lincoln--notebook_24_2017.png)

##### B8 PCR Amplification for sequencing:

![](https://static.igem.org/mediawiki/2017/a/a9/T--UNebraska-Lincoln--notebook_25_2017.png)

## Week 10

More sequencing samples for A and B were sent out by Jessica. Nic performed a [restriction digest](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) of the H insert for restriction digest cloning. The sequencing samples for the B8 colony proved to be very promising so Jessica performed a [protein purification](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) and [western blot](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) on the B8 colony that did not prove successful. This was deemed to be because the protein was producing at its natural level of expression which was very low. Our team decided that it needed to be cloned into an inducible vector.

##### Failed couple digestion using XbaI and SpeI\*:

![](https://static.igem.org/mediawiki/2017/a/ac/T--UNebraska-Lincoln--notebook_26_2017.png)

##### Protein purification of B8:

![](https://static.igem.org/mediawiki/2017/4/4c/T--UNebraska-Lincoln--notebook_27_2017.png)

##### Western Blot of B8 protein purification:

![](https://static.igem.org/mediawiki/2017/b/b4/T--UNebraska-Lincoln--notebook_28_2017.png)

##### Same Western Blot after washing in nanopure water overnight:

![](https://static.igem.org/mediawiki/2017/2/21/T--UNebraska-Lincoln--notebook_29_2017.png)

## Week 11

This week marked the start of our school year. A week long break from the lab was taken during this transition.

## Late August/September/October

Jessica performed [protein purifications](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) and an [enzyme assay](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) for B8 while Nick Flaxbeard worked on characterizing A using the [Nessler’s test](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments). Jessica finished sequencing A. The results showed a deletion in the sequence. Our team decided to no longer pursue the characterization of A and instead we worked on completing the interlab study. Nic worked on using [restriction digest cloning](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments) to reclone H. Nic’s restriction digest cloning was successful so Nick Flaxbeard began characterization of H using the [Nessler’s test](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments).

Protein Purification of bromoperoxidase using an inducible promoter:

![](https://static.igem.org/mediawiki/2017/9/95/T--UNebraska-Lincoln--notebook_30_2017.png)

![](https://static.igem.org/mediawiki/2017/f/f2/T--UNebraska-Lincoln--notebook_31_2017.png)

[Week 1](#week1)

June 5 - June 11

[Week 2](#week2)

June 12 - June 18

[Week 3](#week3)

June 19 - June 25

[Week 4](#week4)

June 26 - July 2

[Week 5](#week5)

July 3 - July 9

[Week 6](#week6)

July 10 - July 16

[Week 7](#week7)

July 17 - July 23

[Week 8](#week8)

July 24 - July 30

[Week 9](#week9)

July 31 - August 6

[Week 10](#week10)

August 7 - August 11

[Week 11](#week11)

August 12 - August 18

[Late August/September/October](#months)

# Thanks to Our Sponsors

![](https://static.igem.org/mediawiki/2017/thumb/b/bf/T--UNebraska-Lincoln--sponsors_2017.png/1200px-T--UNebraska-Lincoln--sponsors_2017.png)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:UNebraska-Lincoln/Experiments

# UNL 2017

## Helping reduce methane emissions from livestock

[![](https://static.igem.org/mediawiki/2017/8/86/T--UNebraska-Lincoln--smallLogo_2017.png)](https://2017.igem.org/Team%3AUNebraska-Lincoln)

[Team](https://2017.igem.org/Team%3AUNebraska-Lincoln/TeamLanding)

[Team](https://2017.igem.org/Team%3AUNebraska-Lincoln/Team)
[Collaborations](https://2017.igem.org/Team%3AUNebraska-Lincoln/Collaborations)

[Project](https://2017.igem.org/Team%3AUNebraska-Lincoln/ProjectLanding)

[Description](https://2017.igem.org/Team%3AUNebraska-Lincoln/Description)
[Design](https://2017.igem.org/Team%3AUNebraska-Lincoln/Design)
[Experiments](https://2017.igem.org/Team%3AUNebraska-Lincoln/Experiments)
[Notebook](https://2017.igem.org/Team%3AUNebraska-Lincoln/Notebook)
[InterLab](https://2017.igem.org/Team%3AUNebraska-Lincoln/InterLab)
[Contribution](https://2017.igem.org/Team%3AUNebraska-Lincoln/Contribution)
[Model](https://2017.igem.org/Team%3AUNebraska-Lincoln/Model)
[Results](https://2017.igem.org/Team%3AUNebraska-Lincoln/Results)
[Attributions](https://2017.igem.org/Team%3AUNebraska-Lincoln/Attributions)

[Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/PartsLanding)

[Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Parts)
[Basic Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AUNebraska-Lincoln/Composite_Part)

[Safety](https://2017.igem.org/Team%3AUNebraska-Lincoln/Safety)

[Human Practices](https://2017.igem.org/Team%3AUNebraska-Lincoln/HumanLanding)

[Silver HP](https://2017.igem.org/Team%3AUNebraska-Lincoln/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3AUNebraska-Lincoln/HP/Gold_Integrated)

![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)
![](https://static.igem.org/mediawiki/2017/6/6c/T--UNebraska-Lincoln--foreground2017.png)

# EXPERIMENTS

### ›Agar Plate Preparation

#### Materials

* 24 petri dishes
* 500 mL LB Agar
* 500 µL cm34

#### Procedure

1. Microwave 500 mL of LB Agar for 15 min on power level 3.
2. Repeat step 1.
3. Put LB Agar in the 55°C water bath until the temperature of the LB Agar equalizes to 55°C.
4. Pipette 500 µL of antibiotic into the LB Agar and mix well.
5. Label the bottom of the empty petri dishes with the name of the antibiotic.
6. Pour LB Agar into petri dishes. Make sure Agar covers the entire bottom of the dish.
7. Flame the petri dishes.
8. Let them solidify at room temperature and then store in the 4°C.

### ›Cell Lysis and Protein Purification

#### Materials

* 1M K2HPO4
* 1M KH2PO4
* 1M NaCl
* Autoclaved H2O
* 1M Imidazole
* 1.5 mL Eppendorf Tubes
* 20% ethanol
* Laemmli Sample Buffer (Bio Rad)
* Coomassie Brilliant Blue stain (Bio Rad)

#### Procedure

1. Prepare a 5 mL starter culture of *E. coli* and let it incubate at 37°C for 16 hours
2. Transfer 50 µL of the starter culture into 50 mL of LB broth and 50 µL antibiotic
3. Let 50 mL culture incubate at 37°C for 24 hours
4. While waiting for the cultures to grow prepare an SDS page and buffers under the specifications below. Adjust pH range between 7-8

   ##### Lysis Buffer

   ![](https://static.igem.org/mediawiki/2017/2/27/T--UNebraska-Lincoln--protocolTable_1_2017.png)

   ##### 20mM Imidazole

   ![](https://static.igem.org/mediawiki/2017/0/09/T--UNebraska-Lincoln--protocolTable_2_2017.png)

   ##### 50mM imidazole

   ![](https://static.igem.org/mediawiki/2017/b/b3/T--UNebraska-Lincoln--protocolTable_3_2017.png)

   ##### Elution Buffer (500mM imidazole)

   ![](https://static.igem.org/mediawiki/2017/f/f0/T--UNebraska-Lincoln--protocolTable_4_2017.png)
5. Centrifuge culture for 10 min at 23°C at 5,000rpm.
6. Weigh the cell wet mass. Then resuspend by pipetting lysis buffer into the solution in a 1:4 ratio of cell wet mass to volume of lysis buffer.
7. Keep everything on ice for the rest of the procedure.
8. Clean the rod of the sonicator with autoclaved H2O and then ethanol.
9. Insert the rod as close as you can to the bottom of the tube without touching the bottom of the tube.
10. Sonicate the cell suspension for 4 minutes using 10 seconds of sonication and 50 second pause intervals.
11. Take the nanodrop protein concentration at 280 absorbance of the cells after they have been lysed.
12. Dilute the Total Fraction(lysed cells) to make 20 µL of 10 mg/mL solution. Set this aside for the SDS page.
13. Centrifuge the rest of the Total for 20 min at max speed at 4°C.
14. While waiting for the centrifuge equilibrate the column by using a 5:1 ration of water to column in terms of volume.
15. Slowly pipette this volume of water onto the edge so the column is not disturbed.
16. Pipette into the column a 3:1 ratio of lysis buffer to column in terms of volume.
17. Once the centrifuge is finished dilute the Total Soluble to make 20 µL of 10 mg/ml solution. Set this aside for the SDS page.
18. Run all of the Total Soluble fraction through the column and collect the Flow Through in 1.5 mL eppendorf tubes.
19. Run 2 mL of the 20mM imidazole wash buffer through the column and collect in 1.5 mL eppendorf tubes.
20. Run 2 mL of the 50mM imidazole wash buffer through the column and collect in 1.5 mL eppendorf tubes.
21. Run 2 mL of the Elution Buffer through the column and collect in 1.5 mL eppendorf tubes.
22. Dilute the flow through tubes to make 20 µL of 10 mg/mL solution. Set this aside for the SDS page.
23. Take 20 µL from each of the leftover from purification and set it aside for the SDS page.
24. Store leftover samples in the -20°C freezer.

    To prepare solutions for the SDS page, make mixtures shown in the table below.

    ![](https://static.igem.org/mediawiki/2017/1/19/T--UNebraska-Lincoln--protocolTable_5_2017.png)
25. Boil tubes in Thermal Cycler for 10 min.
26. Centrifuge tubes to get rid of bubbles.
27. Load wells of the SDS page with 15 µL of each solution.
28. Fill well farthest to the left and farthest to the right with 5 µL of ladder.
29. Run the gel for 30 min at 50 amps.
30. Make sure that gel ran very close to the bottom (but not off the gel), and then wash the SDS page in water.
31. Microwave the SDS page for 10 seconds.
32. Rock until the blue line at the bottom disappears then stain the gel with coomassie blue, microwave for ten more seconds and cover with tinfoil.
33. Let gel stain overnight and take an image in the morning.

### ›Colony PCR

#### Materials

* PCR tubes
* Autoclaved Water
* Cm34 Agar plate
* Taq DNA polymerase
* Primers
* Thermocycler

#### Procedure

1. Fill 8 PCR tubes with 10 µL of autoclaved.
2. Get an unused cm34 agar plate and label the back with the numbers 1-8.
3. Take the pipette and swipe up one of the colonies you want to analyze and then gently swipe the pipette tip onto the empty plate on top of the corresponding number. Be careful not to swipe all of the colony onto the plate. Repeat this for all eight colonies.
4. Transfer the colony into the PCR tube. Pipette up and down to resuspend the cells.
5. Boil the PCR tubes for 10 minutes in the thermocycler.
6. Create a taq DNA polymerase mixture in a 1.5 mL eppendorf tube as such.

   ![](https://static.igem.org/mediawiki/2017/a/a0/T--UNebraska-Lincoln--protocolTable_14_2017.png)
7. Take 1 µL of the first boiled sample and put it into a fresh PCR tube.
8. Pipette 25 µL of the Taq DNA polymerase mix into the fresh PCR tube. Do this for the next eight colonies.
9. Start the Polymerase Chain Reaction in the thermocycler using the specifications defined below.

   ![](https://static.igem.org/mediawiki/2017/a/a4/T--UNebraska-Lincoln--protocolTable_15_2017.png)
10. Load the samples into a gel and perform gel electrophoresis as defined by the protocol to test if the PCR was successful.

### ›Enzymatic Assay of Bromoperoxidase

#### Materials

* MOPS Buffer
* NaOH
* Ethanol
* Monochlorodimedone
* Potassium Bromide
* Hydrogen Peroxide
* Cell Lysate Solution

#### Procedure

The entire procedure should be performed at 25°C with all reagents at pH 6.4.

1. Prepare reagents A-E as Specified below.
   * 50 mM MOPS Buffer, pH 6.4 at 25°C (Prepare 250 ml in deionized water using MOPS, free acid, hydrate, and adjust to pH 6.4 at 25°C with 1 M NaOH.)
   * 100% (v/v) Ethanol (Use Ethyl Alcohol, Anhydrous, 200 Proof)
   * 0.1 mM Monochlorodimedone with 200 mM Potassium Bromide (MCD) (Prepare by dissolving 1.2 gram of Potassium Bromide, in 50 ml of Reagent A. Then dissolve 21.75 mg of Monochlorodimedone, in 0.5 ml of Reagent B. Add 20 µl of the MCD solution to the KBR solution.)
   * 100 mM Hydrogen Peroxide (H2O2) (Prepare by diluting 0.284 ml of Hydrogen Peroxide, 30% (w/w) solution, to 25 ml with Reagent C.)
   * Cell Lysate solution(Incubate at 25°C for 30 minutes prior to use)
2. Pipette (in milliliters) the following reagents into the wells of a UV transparent microplate.

   ![](https://static.igem.org/mediawiki/2017/9/95/Enzyme_protocol.png)
3. With a microplate reader record the decrease in A290nm for several minutes until constant then add 20 µL of hydrogen peroxide to each well to catalyze the reaction.
4. Record the decrease in A290nm for several more minutes. Obtain the A290nm/minute using the maximum linear rate for both the samples and controls.

NOTE= This procedure was adapted from a sigma aldrich protocol. (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General\_Information/2/bromoperoxidase.pdf) We used a microplate reader instead of cuvettes and a spectrophotometer to measure change in absorbance. Because of this 400 µL total instead of 1 mL total. Ratios of reagents are the same just modified to be 400 µL total volume.

### ›Gel DNA Recovery

#### Materials

* Zymoclean™ Gel DNA Recovery Kit
* ADB Buffer
* Wash Buffer

#### Procedure

1. Weigh the gel, then add 3 volumes of ADB buffer to each volume of gel. (max volume in tube = 750 µL)
2. Incubate the 1.5 mL eppendorf tube in a 55°C water bath for 5-10 minutes.
3. Use vortex to mix well.
4. Add the melted agarose solution into a Zymo-spin column and place into a collection tube.
5. Centrifuge for 30 seconds at 16,000 g. Empty the collection tube whenever necessary.
6. Add 200 µL of wash buffer to the column and centrifuge for 30 seconds.
7. Repeat step six.
8. Centrifuge at max speed for 2 minutes.
9. Place Zymo-Spin column into a new 1.5 mL tube.
10. Add 6-10 µL of autoclaved H2O directly to the column matrix.
11. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.

### ›Gel Electrophoresis

#### Materials

* Agarose powder
* TAE buffer
* Erlenmeyer flask
* Ethidium Bromide
* Casting tray
* Sub-Cell apparatus
* 1kb ladder
* 6x Loading Dye

#### Procedure

1. Weigh 0.5 grams of agarose powder.
2. Measure 50 mL of TAE Buffer into an erlenmeyer flask and add in the agarose powder. Mix well.
3. Cover the erlenmeyer flask and microwave for two minutes at power level ten.
4. Let the solution cool for ~10 minutes.
5. Add 4 µL of ethidium bromide and mix well.
6. Pour the solution into a casting tray and let it cool for 30 minutes.
7. Set up the Sub-Cell apparatus and fill with enough TAE buffer to cover the gel.
8. Remove the comb.
9. Load 5 µL of the 1 kb ladder into the leftmost well.
10. Add 5 µL of 10x loading dye into your sample and then load this into a well.
11. Once your samples are loaded place the lid on the apparatus and run the gel at 100 volts for 1 hour.

### ›LB Agar Preparation

#### Materials

* LB Broth powder (Lennox)
* 500 mL media bottle
* LB Agar powder
* 500 mL nanopure water

#### Procedure

1. Weigh 12.5 grams of LB Broth powder and transfer to the 500 mL media bottle.
2. Weigh 7.5 grams of LB Agar powder and transfer to the 500 mL media bottle.
3. Add 500 mL of nanopure water into the media bottle.
4. Mix well.
5. Autoclave media bottle on cycle 2.

### ›LB Broth Preparation

#### Materials

* LB Broth powder (Lennox)
* 250 mL media bottle
* 250 mL nanopure water

#### Procedure

1. Weigh 6.25 grams of LB Broth powder.
2. Add LB Broth powder into a 250 mL media bottle.
3. Pour 250 mL of nanopure water into the media bottle.
4. Mix well.
5. Autoclave media bottle on cycle 2.

### ›Nitrite Metabolism Testing Procedure

Note: In order to simplify the procedure, the symbol N will be used to designate the total number of samples being tested. For example, if five different nitrite concentrations are being tested, N for that experiment would be 12, because there would be five samples containing the various concentrations for both the control and experimental cultures, along with a 0 mmol samples for both cultures. For clarity see Table 1 below:

![](https://static.igem.org/mediawiki/2017/6/63/T--UNebraska-Lincoln--lastMinute_2017.png)

#### Materials

##### Day 1

* Solid NaNO2 (Total required mass is calculated for experiment conditions)
* 150 mL Erlenmeyer Flask
* 50 mL Graduated Cylinder
* LB Media
* 20% Aqueous Glucose
* Serum Vials, Rubber Stoppers, and Metal Caps
* Serum Vial Crimper
* Nitrogen Source and Tube
* 1 mL Syringe Needles
* Ring Stand, Clamps
* Four Culture Tubes
* Chloramphenicol (1 ug/mg)

##### Day 2

* 96 Well Plate
* Chloramphenicol (1 ug/mg)
* Syringes and Needles

##### Day 3

* Syringes
* Nessler’s Reagent

#### Procedure

##### Day 1

###### Nitrite Solution Preparation

1. In separate eppendorf tubes, prepare 100 µL of sodium nitrite solution for each concentration that will be tested in the experiment. The final volume of each anaerobic culture, including the added nitrite solution, will be 10.6 mL.

###### Vial Preparation

1. In a 150 mL Erlenmeyer flask, combine 146.25 mL of LB media with 3.75 mL of 20% aqueous glucose in sterile conditions. LB media can be measured using a 50 mL graduated cylinder.
2. Obtain N of each of serum vials, rubber stoppers, and metal caps
3. Transfer 10 mL of the previously prepared culture into each vial, along with 100µL of the corresponding nitrite solution.
4. Seal each vial with a rubber stopper.
5. Using a vial crimper, crimp a metal cap onto each vial in order to seal the vials shut. Make sure to label this cap to keep track of the nitrite concentration present.
6. Dispose of the remaining media in the Erlenmeyer flask accordingly.

###### Sparging

1. Obtain two 1 mL syringe needles, a ring stand, and two clamps. The syringes themselves are not necessary, only the needles.
2. Fix one clamp onto the ring stand towards the base. Put a serum vial in the gripping end of the clamp and tighten. Make sure the base of the vial is resting on the base of the stand.
3. Mount the other clamp higher on the ring stand. This will hold the nitrogen tube.
4. Attach one of the syringe needles to a nitrogen line. This attachment will vary depending on the nitrogen source setup present in each lab.
5. Use the other syringe needle to vent the serum vial. This needle is perforated through the rubber stopper. DO NOT PUT THE TIP OF THIS NEEDLE INTO THE MEDIA PRESENT IN THE VIAL. This will lead to expulsion of media from the vial onto the surroundings.
6. The serum vial is now vented and securely mounted to the table, and a syringe needle is attached to the nitrogen tube. Slowly begin to turn on the nitrogen source until the desired flow rate is reached. Very little flow is required for sparging.
   After the desired flow is reached, push the nitrogen needle into the vial. Put this tip as far into the liquid as possible so it will bubble through the media. Adjust flow as necessary until a steady stream of bubbles is present.
7. Each vial needs to be sparged for ten minutes. At the end of each sparging, remove the nitrogen source needle first, and then the vent needle. When starting a new vial, insert the vent needle before the nitrogen needle.
8. After all vials have been sparged, shut off the nitrogen supply, dispose of the needles in the sharps container, and disassemble the rest of the setup.
9. After sparging, autoclave the vials using the liquid cycle to ensure any potential contaminants are destroyed.

###### Creating Aerobic Cultures

1. Obtain four culture tubes for this portion of the experiment.
2. In sterile conditions, add 5 mL of LB media with 5 uL of chloramphenicol (1 µg/mg).
3. The first two tubes are both for control cultures. Transfer a single colony from a culture plate into each tube, again in sterile conditions.
4. Repeat the previous step for the experimental cultures.
5. Incubate these cultures overnight in a 37℃ shaker incubator.

##### Day 2

###### Optical Density of Aerobic Cultures

1. Obtain an OD reading for all four aerobic cultures. Blank with water. A 10:1 dilution is recommended, but other dilution factors are acceptable as long as performed consistently.
2. Select the control and experimental cultures with the higher OD reading to perform the rest of the experiment.

###### Creating Anaerobic Cultures

1. Obtain the control and experimental aerobic culture tubes with the higher OD reading. Add 5.6 µL of chloramphenicol antibiotic to each.
2. Obtain N syringes and syringe needles.
3. Syringe 0.5 mL of culture into each vial using a different syringe for each vial. Make sure to add the correct culture in the correct vial (control vs experimental).
4. Incubate the vials overnight in the 37℃ shaker incubator.

##### Day 3

###### Optical Density of Anaerobic Cultures

1. Obtain N Eppendorf tubes.
2. Extract approximately 1.2 mL of each aerobic culture using a different syringe. Put each of these samples into a separate Eppendorf tube.
3. From each Eppendorf tube, pipet 20 µL of culture into the desired well plate. Perform OD reading in the same fashion as the aerobic cultures.

###### Isolation of Supernatant

1. Centrifuge the Eppendorf tubes made in the previous section at approximately 20000 g for three minutes.
2. Pre-weigh N new large Eppendorf tubes.
3. Transfer 1 mL of supernatant from each sample into the corresponding pre-weighed Eppendorf tube. Discard the remaining supernatant and pellet in a biohazard waste container.

###### Nessler’s Test

1. 0.5 mL of Nessler’s Reagent is required for each supernatant sample. Obtain an adequate amount (0.5 mL \* N) of reagent in a disposable culture tube.
2. Add 0.5 mL of the reagent into each supernatant sample tube. Close the lid and invert 5x for each sample to ensure complete reaction of the reagent with the ammonia present in the sample.
3. Centrifuge the samples at approximately 20000 g for 10 minutes.
4. After centrifuge, remove as much supernatant as possible from each sample using a separate pipet tip and discard in a heavy-metal waste container. Be careful to not remove any precipitate.
5. Centrifuge again highest rpm for 10 minutes.
6. Again, remove any remaining supernatant using a separate pipet tip for each sample and dispose in the heavy metal waste container.
7. Weigh each sample and subtract the initial mass of the pre-weighed Eppendorf tube. Leave in the fume hood for 24 hours, and re-weigh. Continue to re-weigh every 24 hours until the masses stabilize. Record the final stabilized masses and subtract the initial corresponding container masses.
8. Using proper technique, clean up any waste in the appropriate manner. Ensure that all items that came into contact with Nessler’s reagent are disposed of in a heavy metals waste container.

### ›Plasmid Mini-Prep

#### Materials

* Thermo Scientific GeneJET Plasmid Miniprep Kit
* 1.5 mL eppendorf tube x4
* Column and Matrix x2
* Wash buffer
* Zyppy wash buffer
* Lysis buffer
* Neutralization buffer

#### Procedure

1. Centrifuge cell culture for 10 min at 23°C at 5,000rpm.
2. Pour out the LB Broth Supernatant into bleach.
3. Resuspend cell pellet in 600 µL of autoclaved H2O.
4. Transfer 600 µL of the cell pellet into a 1.5 mL eppendorf tube.
5. Add 100 µL of lysis buffer to the eppendorf tube.
6. Invert tube 2-4 times and lyse for 2 minutes.
7. Put 350 µL of neutralization buffer into the tube and continue to invert tube until all of the blue color disappears and a yellow precipitate forms.
8. Centrifuge tube at 21,000g for 5 minutes.
9. Transfer the supernatant into the zymo-spin column.
10. Place the column into a collection tube and centrifuge at 16,000 g for 15 seconds. Discard the flow-through and place the column back into the same collection tube.
11. Add 200 µL of Endo-Wash buffer to the column. Centrifuge at 16,000 g for 15 seconds.
12. Add 400 µL of Zyppy-wash buffer to the column. Centrifuge at 16,000 g for 30 seconds.
13. Centrifuge for two minutes at 21,000 g.
14. Transfer the column into a clean 1.5 mL microcentrifuge tube then add 30 µL of Autoclaved H2O directly to the column matrix.
15. Let it stand for one minute at room temperature.
16. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.

### ›Polymerase Chain Reaction

#### Materials

* PCR Tubes
* DNTP’s
* 10x PCR buffer
* MgSO4
* Template DNA
* Primers
* KOD Polymerase
* Autoclaved Water
* Thermocycler

#### Procedure

1. For one 25 mL reaction mix the materials stated below into a PCR tube.

   ![](https://static.igem.org/mediawiki/2017/b/bc/T--UNebraska-Lincoln--change_protocolTable_16_2017.png)
2. Place the reaction tube into the thermocycler and set it based on the specifications below.

   ![](https://static.igem.org/mediawiki/2017/c/cb/T--UNebraska-Lincoln--protocolTable_17_2017.png)

### ›Restriction Enzyme Digest

#### Materials

* Restriction enzymes
* Cutsmart Buffer
* DNA insert
* Plasmid

#### Procedure

1. Prepare 14 µL of uncut DNA insert or plasmid in PCR tube.
2. Add 2 µL of Cutsmart buffer.
3. Add 2 µL of each desired restriction enzyme (i.e. XbaI and SpeI).
4. Gently mix together.
5. Place in thermocycler for 1½ hours at 38°C.
6. Run gel electrophoresis and perform gel DNA recovery on product.
7. Store in 4°C chamber.

### ›Restriction Enzyme Digest Cloning

#### Materials

* T4 Ligase
* Ligase buffer
* Restriction enzymes
* PCR tubes
* Autoclaved Water

#### Procedure

1. Perform restriction enzyme digest on vector using EcoRI and PstI.
2. Perform restriction enzyme digest on DNA insert using XbaI and SpeI.
3. Calculate the amount of cut vector and insert needed to have a 1:3 molar ratio. The total volume of reaction should have a DNA concentration near 10 ng/µL.
4. Load the cut vector and insert into PCR tube.
5. Add enough 10x Ligase Buffer so that the buffer is one-tenth the total volume.
6. Add 0.5 µL of T4 DNA Ligase into the PCR tube.
7. Prepare a negative control by replacing the cut DNA insert portion with water.
8. Place PCR tubes into thermocycler and run the ligation protocol.
9. Transform product.

### ›SDS Page

#### Materials

* 30% acrylamide mixture (29:1)
* 50 mL Flask
* 1.5 M Tris, pH 8.8
* Nanopure Water
* Degassing apparatus
* 10% SDS
* 10% APS
* TEMED
* Gel Casting Module (Bio-Rad)
* 2-propanol
* 1.5 M Tris, pH 6.8

#### Procedure

1. In a 50 mL filter flask, mix the following components for preparing two resolving gel

   ![](https://static.igem.org/mediawiki/2017/f/f3/T--UNebraska-Lincoln--protocolTable_10_2017.png)
2. Degas the solution for at least 30 minutes then add

   ![](https://static.igem.org/mediawiki/2017/3/39/T--UNebraska-Lincoln--protocolTable_11_2017.png)
3. While the gel mixture is degassing, assemble the gel-casting module. Examine the glass plates, the inside of the plate (the side that faces the gel) needs to be free of stain, fiber, or any types of visible particles. Pair a spacer plate with a short plate, and fix them in the casting frame. Make sure the bottoms of the two glass plates are leveled.
4. Examine the rubber seal at the bottom of the casting stand. It should be free of residual gels. Fix the casting frame on the gel casting stand.
5. Place the gel comb between the glass plate. Make a mark on the place about 0.5 cm below the tip of the comb.
6. Take the comb out.
7. Cast the gel by pipetting the resolving gel mixture between the glass plates up the marked line.
8. Add 2-propanol on the top of the gel, let it stand for 1 hour. When waiting start to degass the stacking gel solution.
9. In a 50 mL filter flask, mix the following components for preparing two stacking gels.

   ![](https://static.igem.org/mediawiki/2017/9/9a/T--UNebraska-Lincoln--protocolTable_12_2017.png)
10. Degas the solution for at least 30 min, then add

    ![](https://static.igem.org/mediawiki/2017/c/ce/T--UNebraska-Lincoln--protocolTable_13_2017.png)
11. When the polymerization of the resolving gel is finished, rinse off the alcohol with Nanopure water. Remove liquid between the portion of the plates that is above the resolving gel.
12. Insert the comb between the plates, cast the stacking gel, and let it stand for 1 hour.
13. Remove the comb, rinse it with Deionized Water to remove gel residues. Then rinse it with nanopure water, dry and store it.

### ›SLIC

#### Materials

* Vector Backbone
* Insert
* 10x Buffer 2.1 NEB
* T4 DNA Polymerase
* Autoclaved water
* Thermocycler
* PCR tubes
* Pipets

#### Procedure

1. 150 ng of vector backbone required. Calculate the volume of vector needed for reaction.
2. Calculate the amount of insert needed for reaction based on the vector (1:1 molar ratio).
3. Calculate the amount of autoclaved water needed to bring the final volume to 20 µL and load into a PCR tube.
4. Load 2 µL of 10x Buffer 2.1 into the PCR tube.
5. Load the vector and insert into the PCR tube.
6. Dilute 2 µL of T4 DNA Polymerase into 10 µL of 10x Buffer 2.1.
7. Load 1 µL of the diluted T4 DNA Polymerase into the PCR tube.
8. Place PCR tube into the thermocycler and run SLIC Protocol.
9. Transform product.

   ![](https://static.igem.org/mediawiki/2017/b/ba/T--UNebraska-Lincoln--protocolTable_18_2017.png)

### ›Transformation

#### Materials

* Genehog Chemically Competent Cells
* Water Bath
* Eppendorf Tubes
* Chloramphenicol Agar Plates
* Micropipet
* Incubator (shaker)
* Sterilized Beads

#### Procedure

1. Thaw 40 µL of competent cells on ice for 5-10 minutes.
2. Add about 100 ng of DNA then continue icing for 20-30 minutes. The added volume should be less than 10% of total volume.
3. Place cells into 42°C water-bath for 35 seconds.
4. Put cells back on ice for 2 minutes. Sterilize work area and pipet in this time.
5. Add 200µL of LB broth to the mixture.
6. Leave the cells at 37°C for 45-60 minutes in shaker. Meantime, prewarm agar plates.
7. Plate cells using beads (volume varies slightly).
8. Incubate the plate overnight at 37°C.

### ›Western Blot

#### Materials

* Bio-Rad Blotting-Grade Blocker
* Methanol
* Nanopure Water
* Tris
* 1 x PBST Buffer
* Bovine Serum Albumin (BSA)
* Nitrocellulose membrane (Bio-Rad)
* Western Blotting Filter Paper (Bio-Rad)
* Ice packs
* Stir Bar
* Mini Trans Blot Cell (Bio-Rad)
* Primary Antibody
* Secondary Antibody
* Opti-4CN diluent concentration
* Opti-4CN Substrate

#### Procedure

![](https://static.igem.org/mediawiki/2017/7/73/T--UNebraska-Lincoln--protocolTable_6_2017.png)

![](https://static.igem.org/mediawiki/2017/4/4b/T--UNebraska-Lincoln--protocolTable_7_2017.png)

![](https://static.igem.org/mediawiki/2017/e/e2/T--UNebraska-Lincoln--protocolTable_8_2017.png)

1. Soak the gel in the Transfer Buffer for 10-15 minutes.
2. Assemble the transfer sandwich and make sure no air bubbles are trapped in the sandwich. The blot should be on the cathode and the gel on the anode.
3. Order of sandwich.
   1. Matrix
   2. Filter Paper
   3. Membrane
   4. Gel
   5. Filter Paper
   6. Matrix
4. To set up the apparatus, place the cassette in the transfer tank. Fill up to the four gel line with Transfer Buffer. Add a stir bar and stir the solution. Add ice packs to keep it cool.
5. Run for one hour at 100 volts.
6. Disassemble the apparatus, inspect the membrane and the PAGE. Except the largest size band, all other marker bands should be invisible. Air-dry the membrane for ~ 5 min.
7. Wash for 5 min in 1 x PBST buffer. (40 cm2 blot, 10 mL/wash; 60 cm2 blot, 15 mL/wash) and put on a rocker.
8. Block for one hour with Blocking Solution.
9. Wash for 5 min in 1 x PBST buffer. (40 cm2 blot, 10 mL/wash; 60 cm2 blot, 15 mL/wash) and put on a rocker.
10. Blot with primary antibody in 6 mL 1 x PBST with 1% BSA for 1 h.
11. Wash for 5 min in 1 x PBST buffer. (40 cm2 blot, 10 mL/wash; 60 cm2 blot, 15 mL/wash) and put on a rocker.
12. Blot with secondary antibody in 6 mL 1 x PBST with 1% BSA for 1 h.
13. Wash for 5 min in 1 x PBST buffer. (40 cm2 blot, 10 mL/wash; 60 cm2 blot, 15 mL/wash) and put on a rocker.
14. Prepare the detection solution. Make sure you use it as quickly as possible.

    ![](https://static.igem.org/mediawiki/2017/4/4f/T--UNebraska-Lincoln--protocolTable_9_2017.png)
15. Add the solution to blot and incubate for 1 minutes or more to obtain desired result.
16. Pour off detection solution, wash membrane in nanopure H2O for 15 min.
17. Document results.

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# Team:UNebraska-Lincoln/Results

# UNL 2017

## Helping reduce methane emissions from livestock

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# Results

### Overview

We are excited to present the following results:

* Successfully cloned and fully sequenced without mutation two new biobrick parts. A new nitrite reductase enzyme and vanadium-dependent bromoperoxidase.
* Observed potential increase in nitrite metabolism for the nrfAH plasmid at concentrations around 30 mM NaNO2 with respect to the empty vector.
* Proved that our bromoperoxidase protein is being produced and works as expected.

## Nitrite Reductase

### Background

One of the potential ways to reduce methanogenesis in cattle is to divert resources away from methanogens to other microorganisms in the gut. Nitrogen-fixing bacteria present in the rumen compete with methanogens for hydrogen, using it to make ammonium which can then be rapidly converted to amino acids by other microorganisms. The reduction route from nitrate to ammonium is shown in the following equation.

![](https://static.igem.org/mediawiki/2017/e/eb/T--UNebraska-Lincoln--resultsPic_1_2017.png)

In comparison to the reduction of nitrate to nitrite, reduction of nitrite to ammonium is relatively slower. Because the nitrite anion is substantially more toxic than the other molecular species in the nitrate reduction pathway, elimination of nitrite is the key step. To solve this problem, nitrogen-fixing bacteria containing a gene that encodes a more efficient nitrite reductase is required in the rumen. As a consequence, methanogenesis could be reduced, and protein production could increase for the animal as well.

To achieve this goal, we transformed a plasmid encoding a nitrite-reductase enzyme into an *E. coli* chassis. Once a successfully-transformed strain was obtained, we then examined the cell growth and nitrite reduction activity in conditions similar to the cattle rumen.

Nitrite reductase activity was analyzed using Nessler’s Test. Nessler’s Test is a quantitative chemical reaction that is used to determine ammonium concentration in solution. The amount of ammonium present is directly correlated with nitrite reductase activity. Nessler’s Reagent contains 0.09M potassium tetraiodomercurate (K2[HgI4]) in 2.5 M aqueous potassium hydroxide solution. The reaction of this reagent with aqueous ammonium is shown in the following equation.

![](https://static.igem.org/mediawiki/2017/8/80/T--UNebraska-Lincoln--newNewResults_2017.pdf)

### Testing

* Successfully introduce a plasmid containing a nitrite reductase gene into an *E. coli* chassis.
* Grow the transformed *E. coli* under aerobic condition as a seed culture. A strain containing a plasmid encoding RFP (red fluorescence protein) was used as the control for the characterization of NrfA (*E. coli* Cytochrome C nitrite reductase). A culture containing the empty vector is used as a control in the characterization of NrfHA (*S. ruminantium* Cytochrome C nitrite reductase).
* Inoculate the anaerobic vials with seed culture. Media contained varying concentrations of nitrite. Cells were cultured overnight.
* Take OD measurements of the cultures at 600 nm to monitor cell growth.
* Collect cell-free supernatant and test ammonium concentration using Nessler’s Reagent. (Nessler’s reagent reacts with ammonium in solution to form a brown precipitate. The mass of this precipitate is used to gauge the nitrite metabolism.)

### Data Analysis (Rounds 1-2):

*E. coli* strain containing the RFP (red fluorescence protein) plasmid was used for the control in rounds 1-2, while our experimental strain contained a defective form of the nrfA plasmid. The cell chassis used for both strains was Genehog. Average optical density readings (n=4) for the aerobic seed cultures for rounds 1-2 are shown below after 24 hours of growth in Figure 1. Both strains grew well under aerobic conditions. The control strain, on average, grew much faster than the experimental one.

![](https://static.igem.org/mediawiki/2017/1/17/T--UNebraska-Lincoln--newNewResults_1_2017.pdf)

Average optical density (n=2) of anaerobic culture for rounds 1-2 after 24 hours of growth. The OD600nm are plotted against nitrite concentration below in Figure 2. For the control strain, growth is consistent in media containing 0-8 mM nitrite, but drops substantially at 16 mM. For the experimental strain, growth is consistent in media containing 0-4 mM nitrite, but drops for concentrations above 8 mM. However, the growth of the experimental strain is slightly better than the growth of the control strain at 16 mM nitrite.

![](https://static.igem.org/mediawiki/2017/8/8d/T--UNebraska-Lincoln--newNewResults_2_2017.pdf)

Nessler’s Test was performed (n=2) on the cell-free supernatant of each culture. Averaged data for rounds 1-2 is displayed below for each nitrite concentration along with standard deviation in Figure 3. Precipitate mass from Nessler’s Test serves as an indicator of ammonium present in solution. Precipitate mass for both the control and experimental cultures steadily increased as nitrite concentration increased, up to 8 mM nitrite. This likely indicates that both strains produce ammonium. However, at 16 mM, the control strain had largely died off and was unable to metabolize any nitrite, while the experimental strain was still performing adequately. Sequencing results obtained after the completion of round 2 showed a deletion in the open reading frame of the nrfA gene in the experimental strain. The better growth by the experimental strain at 16 mM nitrite is likely due to the decreased metabolic burden with respect to the control, as it did not have to overexpress a protein. The higher concentration of ammonium accumulation by the experimental strain at 16 mM is likely due to the *E. coli* native nitrite reductase activity under the anaerobic culturing condition.

![](https://static.igem.org/mediawiki/2017/7/74/T--UNebraska-Lincoln--newNewResults_3_2017.pdf)

### Data Analysis (Round 3):

*E. coli* strain containing the empty vector (pSB1C3) was used for the control in round 3, while our experimental strain contained the nrfHA plasmid. The cell chassis used for both strains was DH5α, which showed better transformation efficiency. Due to time constraints, we were only able to characterize the new nrfHA strains once.

Average optical density readings (n=2) for the aerobic seed cultures after 24 hours of growth for round three are shown below in Figure 4. Experimental and control strains had comparable growth under aerobic conditions.

![](https://static.igem.org/mediawiki/2017/1/1e/T--UNebraska-Lincoln--newNewResults_4_2017.pdf)

Optical density of anaerobic cultures (n=1) for round three after 24 hours of growth are plotted against nitrite concentration in the media in Figure 5. A much larger range of nitrite concentrations (0-50 mM) were examined in this round. The control grew significantly faster than the experimental strain in the 0 mM nitrite media. This suggests that the difference in metabolic burden caused by production of the nitrite reductase enzyme may be exacerbated by the anaerobic conditions. However, only one experiment was performed with these strains, so additional data would need to be obtained to confirm the hypothesis. In media containing nitrite (10-50 mM), both strains had poor growth, and optical densities were very low across the board. This suggests that neither strain could survive in the tested nitrite concentrations.

![](https://static.igem.org/mediawiki/2017/9/9e/T--UNebraska-Lincoln--newNewResults_5_2017.pdf)

Nessler’s Test was performed (n=1) on the cell-free supernatant of each culture. Precipitate data for round 3 is displayed below for each nitrite concentration in Figure 6. Nessler’s Test data largely paralleled the optical density measurements for the anaerobic cultures. The relatively large precipitate mass present in the control strain at 0 mM suggests that *E. coli* produces ammonium as a byproduct of its own metabolism. This conclusion is supported by the first two rounds of experimentation as well. For the rest of the cultures, precipitate mass was extremely low, with the small spike at 30 mM nitrite in the experimental strain, which can either be explained by experimental error or a potential increase in nitrite reductase activity. Again, more data is needed to clarify this observation.

![](https://static.igem.org/mediawiki/2017/d/dc/T--UNebraska-Lincoln--newNewResults_6_2017.pdf)

## Bromoperoxidase

### Background

Seaweed is now being studied as a food additive because it has been shown to reduce the amount of methane produced by cattle. The compound within seaweed that is responsible for reducing methane is bromoform. Bromoform works by inhibiting the efficiency of the methyltransferase enzyme by reacting with the reduced vitamin B12 cofactor required for the second to last step of methanogenesis. We set out to successfully insert a plasmid containing a bromoperoxidase enzyme from the algae corallina *pilulifera* into an *E. coli* chassis. Once a successfully transformed strain was produced, we could then begin testing how it would fare in conditions similar to the rumen of a cow.

### Testing

* Successfully introduce a plasmid containing the bromoperoxidase gene into an *E. coli* chassis.
* Perform protein purification procedures to isolate the bromoperoxidase enzyme.
* Use the isolated enzyme in the monochlorodimedone enzyme assay.
* Mix the isolated enzyme with potassium bromide, monochlorodimedone and the MOPS buffer in a UV Transparent plate reader.
* Record the absorbance at 290 and ensure that it is constant.
* Add hydrogen peroxide to catalyze the reaction and record the decrease in absorbance at 290.

### Data Analysis

After a couple of attempts we successfully cloned bromoperoxidase into the pSB1C3 backbone. We had an inclination that the eighth colony on the bromoperoxidase agar plate was cloned correctly when we performed PCR with primers that replicated at the prefix and suffix and obtained the results shown below.

![](https://static.igem.org/mediawiki/2017/0/04/T--UNebraska-Lincoln--resultsPic_9_2017.png)

Under the B8 label two bright bands can be seen. This was a good sign that our gene insert was successfully cloned into our *E. coli*. We used the iGEM primers VF2 and VR that were designed to replicate at the prefix and suffix site. After we obtained a thick bright band as a result we knew that the prefix and suffix sites were present in our plasmid. The next step was to sequence our plasmid to ensure that it was cloned without deletions or mutations. We spent the next week or so fully sequencing the plasmid. The sequencing results were positive and ensured us that the bromoperoxidase gene was successfully cloned into our *E. coli* without mutations.

After receiving this great news we immediately started protein purifications to see if the bromoperoxidase protein was being produced. If the *E. coli* is producing the bromoperoxidase protein then a band should be seen at 64 kDa. The resulting SDS Page is shown below.

![](https://static.igem.org/mediawiki/2017/3/37/T--UNebraska-Lincoln--resultsPic_10_2017.png)

There is a small band present in the 500 mM imidazole wash at approximately 64 kDa. This indicates that a small amount of enzyme is being produced. This was a positive sign. Due to our constitutive promoter we were worried that our protein might not be expressed at a noticeable rate. We would suggest that if any other team continues this experiment they should start from the beginning with an inducible promoter. This will help later with characterizing the part. With the constitutive promoter only a small amount of the enzyme was being produced at its natural expression rate.

Next we performed the monochlorodimedone enzyme assay. We used cell lysate first and a control composed of bromide ions, monochlorodimedone and a buffer, but no cell lysate. We catalyzed both solutions with hydrogen peroxide according to the protocol.

![](https://static.igem.org/mediawiki/2017/b/be/T--UNebraska-Lincoln--resTable_1_2017.png)
![](https://static.igem.org/mediawiki/2017/a/a8/T--UNebraska-Lincoln--resTable_2_2017.png)

If the monochlorodimedone is being brominated by our enzyme then the absorbance at 290 should decrease. This is exactly what we found.

![](https://static.igem.org/mediawiki/2017/a/a7/T--UNebraska-Lincoln--resultsPic_11_2017.png)
![](https://static.igem.org/mediawiki/2017/0/00/T--UNebraska-Lincoln--resultsPic_12_2017.png)

The results above indicate that the enzyme is performing its designed function because monochlorodimedone is being brominated. This is shown by the decrease in absorbance in the cell lysate compared to the decrease in absorbance of the control solution. Although both the control and the cell lysate show a decrease in absorbance, the cell lysate has a steeper slope indicating greater absorbance. The data points for the cell lysate also had a higher R2 value of .889 compared to the R2 value of .195 (control). This shows that the data points from the cell lysate are significantly closer to the fitted regression line. This is a good sign that our data is following a trend and decreasing while the control seems to be increasing and decreasing randomly.

The next step in analyzing the data is to determine how many units of monochlorodimedone can be brominated per milliliter of enzyme. One unit will catalyze the conversion of 1.0 micromole of monochlorodimedone to monobromochlorodimedone per min at pH 6.4 at 25°C. To do this use the equation shown below

![](https://static.igem.org/mediawiki/2017/1/1a/T--UNebraska-Lincoln--resultsPic_13_2017.png)
![](https://static.igem.org/mediawiki/2017/a/a9/T--UNebraska-Lincoln--resTable_3_2017.png)

After this calculation was done the cell lysate was found to be converting 0.0043 units of monochlorodimedone per milliliter of enzyme. This proves that the enzyme is working as expected. To perform further characterization more assays were performed.

Next the cell lysate containing bromoperoxidase was tested again to ensure that the results still showed a decrease in absorbance at 290. The cell lysate was compared to the control solution along with a cell lysate containing the empty vector pSB1C3.

![](https://static.igem.org/mediawiki/2017/3/30/T--UNebraska-Lincoln--resTable_4_2017.png)

Both reactions were catalyzed with hydrogen peroxide as referenced in the protocol.

![](https://static.igem.org/mediawiki/2017/e/e8/T--UNebraska-Lincoln--resultsPic_14_2017.png)
![](https://static.igem.org/mediawiki/2017/5/5c/T--UNebraska-Lincoln--resultsPic_15_2017.png)

The above results further display that our part is working as expected. When hydrogen peroxide is added to the cell lysate containing bromoperoxidase there is a negative slope indicating a decrease in absorbance. When hydrogen peroxide is added to the empty vector the slope has a positive value indicating an increase in absorbance. Both cell lysates stick to their regression lines roughly the same amount. The bromoperoxidase cell lysate has a R2 value of 0.693 and the empty vector cell lysate has a R2 value of 0.652. This shows that both reactions followed their trend line to a significant degree. This is good news because it means that our data was not random, instead it was being controlled by the bromination of monochlorodimedone into monobromochlorodimedone.

In this trial the cell lysate was found to be converting 0.00094 units of monochlorodimedone per milliliter of enzyme. The cell lysate with the empty vector was converting -0.0019 units of monochlorodimedone per milliliter of enzyme. This makes sense because the empty vector was not brominating monochlorodimedone.

![](https://static.igem.org/mediawiki/2017/3/36/T--UNebraska-Lincoln--resTable_5_2017.png)

Next we compiled the data from all of our trials using the cell lysate with bromoperoxidase and compared it to the control solution that was used in every trial. Below is a graph showing the results.

![](https://static.igem.org/mediawiki/2017/c/cf/T--UNebraska-Lincoln--resultsPic_16_2017.png)

Statistical analysis shows that the cell lysate from *E. coli* containing the bromoperoxidase plasmid has significant activity. Although the cell lysate showed positive results we wanted to test the purified enzyme. Unfortunately, we were not able to perform a test on the purified enzyme itself, because there were issues with the protein purification method used. We used the Ni-NTA Purification System, but in the literature the DEAE-Sepharose Fast Flow column was used. If another iGEM team were to continue our project we would suggest avoiding the Ni-NTA Purification System and using the DEAE-Sepharose Fast Flow column method. We hypothesize that the His tag purification method did not work well because our enzyme is vanadium dependent. The vanadium ions at the active center act similarly to the immobilized nickel ions present in the resin. Because of this, the nickel ion may interfere with the binding of the vanadium ion in the active site and cause the purified enzyme to lose activity.

[Nitrite Reductase](#nitrite)

[Bromoperoxidase](#bromo)

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