iGEM 2019 — Team OhioState

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# Team:OhioState/Notebook

[![](https://2019.igem.org/wiki/images/7/71/T--OhioState--BlueLogoSmall.png)](https://2019.igem.org/Team%3AOhioState)

* Project

  [Description](https://2019.igem.org/Team%3AOhioState/Description)
  [Design](https://2019.igem.org/Team%3AOhioState/Design)
  [Experiments](https://2019.igem.org/Team%3AOhioState/Experiments)
  [Notebook](https://2019.igem.org/Team%3AOhioState/Notebook)
  [Results](https://2019.igem.org/Team%3AOhioState/Results)
* [Parts](https://2019.igem.org/Team%3AOhioState/Parts)

  [Contribution](https://2019.igem.org/Team%3AOhioState/Contribution)
  [Characterization](https://2019.igem.org/Team%3AOhioState/Measurement)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AOhioState/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3AOhioState/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3AOhioState/Collaborations)
* Judging

  [Judging Form](https://igem.org/2019_Judging_Form?team=OhioState)
  [Safety](https://2019.igem.org/Team%3AOhioState/Safety)
  [Medal Criteria](https://2019.igem.org/Team%3AOhioState/Medal_Criteria)
* Team

  [Team Members](https://2019.igem.org/Team%3AOhioState/Team)
  [Attributions](https://2019.igem.org/Team%3AOhioState/Attributions)

![](https://2019.igem.org/wiki/images/4/47/T--OhioState--NotebookHero.png)

# 6/17-6/21

## Tour Greenhouse Space

**Who?**
Allison, Jacob, Jason

**Protocol:**
N/A

**Notes:**
We met with Dr. Gschwend, a professor in the Dept. of Horticulture and Crop Science, about the possibility of using greenhouse space for testing and growth purposes if we ended up needing a space for controlled growth of transfected plant species.

**Results:**
N/A

## Meeting with Justin North

**Who?**
Allison, Chris, Jason, Jacob, Prajwal

**Protocol:**
N/A

**Notes:**
Dr. Justin North, a post-doc researcher in the Dept. of Microbiology, gave the team an in depth lecture about the structure and function of nitrogenase, as well as challenges and considerations with cloning it.

**Results:**
N/A

## Primer Design

**Who?**
Jason, Jacob, Prajwal

**Protocol:**
N/A

**Notes:**
We started the process of designing primers for our experiments. This included extensive research on the genome of our donor strain (R. palustris) and how to properly piece together the desired parts.

**Results:**
N/A

## Meeting with Erin Gloag

**Who?**
Jacob, Prajwal, Jason

**Protocol:**
N/A

**Notes:**
We met with Dr. Erin Gloag, a post-doc researcher in the Dept. of Microbial Infection and Immunity, about the use of Pseudomonas vectors and recipient strains for our project. She also gave some pointers and tips for Gibson Assembly and some helpful resources to look into.

**Results:**
N/A

# 6/24-6/28

## Streak Out/Culture Initial Strains

**Who?**
Prajwal, Jacob, Jason

**Protocol:**
Bacterial Liquid Culturing, 3-Phase Streak

**Notes:**
We grew up P. protegens PF-5 and R. palustris CGA010 for convenient use later on in the summer

**Results:**
We got healthy cultures and colonies of our desired bacteria.

## Parts Discussion

**Who?**
Prajwal, Jacob, Jason

**Protocol:**
N/A

**Notes:**
We sat in a room and specifically went through all of the different medal requirements regarding lab work and laid out plans of how we intended to fulfill each of them (i.e. Characterization, Part Improvement, and New Part Addition).

**Results:**
Though our plans changed throughout the course of the summer, this meeting helped guide experimental planning and kept us on track throughout the entirety of the project.

## Primer Design

**Who?**
Jacob, Prajwal, Jason

**Protocol:**
N/A

**Notes:**
We continued research on our donor genome, and began to actually draft out primers (using Geneious software).

**Results:**
N/A

## Protocol Design/Evaluation

**Who?**
Jacob, Prajwal, Jason, Chris

**Protocol:**
N/A

**Notes:**
We consulted our supervisors and did a literature search for all of the lab protocols that we would possibly need for the summer. We then put these in a common place (Benchling) so that we could easily reference them whenever needed.

**Results:**
N/A

# 7/1-7/5

## Primer Design

**Who?**
Jacob, Jason, Prajwal

**Protocol:**
N/A

**Notes:**
We finally finished and got an approval on our primers, and how exactly we would piece together our desired part.

**Results:**
Once approved by one of our supervisors, we sent out an order for our primers.

## Literature and Lab Update

**Who?**
Whole Team

**Protocol:**
N/A

**Notes:**
Now that we were about to start the “real” lab work, we all updated ourselves on necessary literature and reviewed our strategies for getting our main project to work and what the next steps were.

**Results:**
N/A

# 7/8- 7/12

## Genome Extraction

**Who?**
Jacob, Prajwal

**Protocol:**
Genomic DNA Extraction

**Notes:**
We headed to Justin North’s lab (where our R. palustris was growing) and extracted gDNA from R. palustris for later use in PCR experiments.

**Results:**
We brought a small microcentrifuge tube back to the lab, which served as our source of gDNA for the next few weeks.

## Plate Mini-CTX1

**Who?**
Prajwal

**Protocol:**
N/A Three-phase streak??

**Notes:**
Prajwal received a plate of Mini-CTX1 from Dr. Erin Gloag (who had a stock lying around) that we planned to use for transformation of our Gibson Assembly product.

**Results:**
We had plentiful colonies that were all healthy and restreaked every few weeks to be kept fresh.

# 7/15-7/19

## Miniprep of CTX-1

**Who?**
Prajwal

**Protocol:**
Liquid Culture Setup, Miniprep

**Notes:**
Prajwal set up a liquid culture (5 mL) of Mini-CTX1, and then performed a miniprep the day after.

**Results:**
This miniprep yielded a DNA concentration of 483.5 ng/uL.

## Primer Resuspension

**Who?**
Prajwal, Jason, Jacob

**Protocol:**
IDT Primer Resuspension

**Notes:**
Once we received our primers in the mail, we performed calculations and then resuspended our primers to usable concentrations for our PCR experiments.

**Results:**
After completing the protocol, we got our primers to practical aliquots at a concentration of 10 uM.

## Initial PCR

**Who?**
Jacob, Jason, Prajwal

**Protocol:**
2-Step PCR

**Notes:**
We performed an initial PCR on our gDNA (this included all 7 parts).

**Results:**
These samples were taken out of the thermocycler after the end of the reaction, and were left to be analyzed on a gel on a different day.

## Gel Electrophoresis

**Who?**
Prajwal, Jason

**Protocol:**
Gel Electrophoresis

**Notes:**
We ran all 7 PCR products on the same gel, let it run, then looked at the gel under a UV lamp for analysis.

**Results:**
Fragment 5 was the only positive result, everything else failed.

# 7/22-7/26

## PCR of Fragment 6

**Who?**
Jason, Prajwal

**Protocol:**
2-Step PCR

**Notes:**
We set up another PCR reaction of just fragment 6, using the exact same protocol from the previous attempt.

**Results:**
After gel analysis, fragment 6 appeared, and the PCR product was preserved.

## Master Mix Design + PCR

**Who?**
Jacob, Prajwal, Jason

**Protocol:**
2-Step PCR

**Notes:**
We decided to reprogram our thermocycler to employ a linear gradient variation in the temperature that the reaction was run at (increasing by a decreased amount each “place” over in the thermocycler). We figured that since we were doing an unusual reaction, it’d be best to find optimal temperatures for the fragments we had. This included fragments 1, 2, 3, 4, and 7

**Results:**
After gel analysis, we got fragments 1, 2, 3, and 7 to work. These products were preserved.

## Fragment 4 Reattempt

**Who?**
Jacob, Jason, Prajwal

**Protocol:**
2-Step PCR

**Notes:**
We decided to make another master mix (for use in the temperature linear gradient scheme) of fragment 4, and then run a PCR of the fragment.

**Results:**
Even with 12 samples at different temperatures, we were unable to get any successful products after running the samples on a gel.

# 7/29-8/2

## Sequencing of Fragments 1, 2, 3, 5, 6 and 7

**Who?**
Jacob, Prajwal

**Protocol:**
N/A

**Notes:**
These fragments were sequenced by an in-house department that just required products to be aliquoted and diluted to a certain concentration.

**Results:**
Once we got sequencing data back, we opened it in Geneious software and learned that we did in fact have the products we wanted, and just needed fragment 4 to begin Gibson Assembly.

# 8/5-8/9

## Fragment 4 Reattempt and Troubleshooting

**Who?**
Jacob, Prajwal

**Protocol:**
2-Step PCR

**Notes:**
We once again tried the same method of PCR for fragment 4. While it was running, we examined our primers for fragment 4 and realized that they were not optimized for our experiment and learned that they had considerable hairpin temperatures.

**Results:**
After another unsuccessful gel, we decided to redesign our primers and immediately order them so that we can move on with our project (due to our design scheme, fragment 4 was a crucial element and the rest of the assembly could not move on without it).

# 8/12-8/16

## Fragment 4 PCR with New Primers and Sequencing

**Who?**
Jacob

**Protocol:**
2-Step PCR

**Notes:**
After receiving our redesigned primers, we redid our PCR of fragment 4 with the linear gradient scheme.

**Results:**
After running a gel, we finally got fragment 4 to work. We purified the PCR product and got it ready for Gibson Assembly.

## Designing Microaerophilic Growth Experiments

**Who?**
Jacob

**Protocol:**
N/A

**Notes:**
Now that we had our products ready for Gibson assembly, we began designing experiments that were supposed to be taking place in microaerophilic environments, and therefore required special precautions and considerations.

**Results:**
N/A

# 8/19-8/23

## Gibson Assembly

**Who?**
Jacob

**Protocol:**
Gibson Assembly

**Notes:**
After re-verifying that we had the right reagents and a usable amount of PCR products we finally attempted our Gibson Assembly of our fragments. We then transformed the final Gibson product into NEB E. coli 10-Beta and left it to grow overnight on LB tetracycline plates.

**Results:**
After overnight growth, we had colonies on the tetracycline plate, indicating that our transformation worked. We now just needed to send the product off for sequence verification.

# 8/24-8/30

## Patching of colonies off Gibson plates to screen for insert

**Who?**
Jacob

**Protocol:**
n/a

**Notes:**
Patching of colonies off of Gibson plates to screen for insert

**Results:**N/A

## 2-Step PCR

**Who?**
Jacob

**Protocol:**
2-Step PCR

**Notes:** Colony PCR of patched colonies using insert verification primers

**Results:** PCR was not successful. Most wells evaporated and the ones that didn’t had reduced product

# 9/3-9/10

## Gel Electrophoresis

**Who?**
Jacob

**Protocol:**
Gel Electrophoresis

**Notes:** Gel of remaining PCR wells (~11)

**Results:** All wells showed a long smear-not interpretable results

## Miniprep

**Who?**
Jacob

**Protocol:**
Miniprep

**Notes:** The plasmids from each of the patched colonies corresponding to the wells run on the gel were miniprepped

**Results:** High DNA concentrations were obtained.

## PCR

**Who?**
Jacob

**Protocol:**
PCR

**Notes:** Redid Colony PCR using Miniprepped plasmids

**Results:** N/A

## Gel Electrophoresis

**Who?**
Jacob

**Protocol:**
Gel Electrophoresis

**Notes:** Gel of plasmid PCR

**Results:** All wells showed a long smear

## Restriction Digest

**Who?**
Jacob

**Protocol:**
Restriction Digest

**Notes:** The plasmids miniprepped out of the patched colonies were digested in order to linearize them

**Results:** N/A

## Gel Electrophoresis

**Who?**
Jacob

**Protocol:**
Gel Electrophoresis

**Notes:** Gel of Minipreps

**Results:** All colonies had a plasmid ~5.5kb, which is the expected size of our vector without the insert.

## Nitrogen Limited Media

**Who?**
Jacob, Prajwal

**Protocol:**
Nitrogen Limited Media

**Notes:** Nitrogen Free, Nitrogen Limited, and Nitrogen Excess media were created for the culturing of diazotrophs (Jensen’s Broth)

**Results:** N/A

## Replica Plating

**Who?**
Jacob

**Protocol:**
Replica Plating

**Notes:** The initial Gibson reaction platings were replica plated onto the nitrogen limited media and left to grow in the anaerobic chamber.

**Results:** After about 1 week a few colonies grew on each the nitrogen free and nitrogen deficient media. These were subcultured into LBTet and grown aerobically.

# 9/12-9/19

## PCR

**Who?**
Jacob

**Protocol:**
PCR

**Notes:** Attempted to use our long range polymerase to amplify the Whole nif cluster in I shot

**Results:** N/A

## Gel Electrophoresis

**Who?**
Jacob, Prajwal

**Protocol:**
Gel Electrophoresis

**Notes:** PCR of whole nif cluster

**Results:** No bands observed

## Three-Phase Streak

**Who?**
Jacob

**Protocol:**
Three-Phase Streak

**Notes:** Pseudomonas Protegens was streaked out onto the nitrogen limited media and left to grow in the anaerobic chamber

**Results:** No growth on Nitrogen free, deficient media, small growth on Nitrogen enriched media after 1 week

# 9/20-9/23

## PCR

**Who?**
Jacob

**Protocol:**
PCR

**Notes:** Colony PCR of colonies from anaerobic chamber

**Results:** N/A

## Gel Electrophoresis

**Who?**
Jacob

**Protocol:**
Gel Electrophoresis

**Notes:** Gel of Colony PCR products

**Results:** No bands observed

# 10/7-10/13

## Miniprep

**Who?**
Jacob

**Protocol:**
Miniprep

**Notes:** Miniprep of colonies isolated from anaerobic chamber

**Results:** Very low DNA concentrations coming out of these cultures; difficult to even visualize these preps on a gel, much less sequence or PCR.

## Filter Mating

**Who?**
Jacob

**Protocol:**
Filter Mating

**Notes:** E.coli+pULB113 (RP4::mini-mu) and R.palustris were mated to try and get the plasmid into R.palustris and fragment its genome.

**Results:** Currently growing in anaerobic chamber to select for successful matings.

# LasR-Dependent Promoter Characterization

# 6/24-6/28:

## Parts Discussion

**Who?**
Jacob, Prajwal, Jason

**Protocol:**
N/A

**Notes:**
We discussed how we were going to approach characterizing a part, which parts we should consider looking into, and how we would go about it.

**Results:**
This discussion guided our experimental planning and activity for the coming months and helped to keep us on track, as well as keep us constantly evaluating whether or not we were doing our best to fulfill medal requirements.

## Chemical Transformation of Biobricks

**Who?**
Jason, Prajwal

**Protocol:**
Mach1 Transformation

**Notes:**
After selecting the biobricks that we wanted to use for experimentation (in this case, BBa\_K575010 from the 2011 Northwestern team), we chemically transformed them to get them ready for further research.

**Results:**
After growing overnight, we got successful colonies of our biobrick-containing plasmid.

# 7/8-7/12

## Electroporations of LasR into E. coli MG1655

**Who?**
Prajwal, Jason

**Protocol:**
Electroporation (Ahmer)

**Notes:**
For initial characterization attempts, we tried to electroporate our biobrick plasmid into MG1655, and then try to induce expression.

**Results:**
After electroporation, we had no successful transformants after overnight growth. We then redid the experiment and had successful colonies that were re-streaked for future use.

## Electroporations of pAL 105/106 into the MG1655-LasR Transformants

**Who?**
Jason, Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
After searching for available options that could assist in the characterization of our biobrick, we turned to our supervisor’s past research and decided to use the pAL105 (LasR +) and pAL106 (LasR -) plasmids.

**Results:**
We only got successful transformants from the pAL106 plasmid to give us a construct that had the LasR-Dependent promoter (with an RFP reporter), but no LasR protein.

# 7/15-7/19, 7/22-7/26

## More Attempts at Transforming pAL105 into the MG1655-LasR Transformants

**Who?**
Prajwal, Jason, Jacob

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We tried multiple attempts and methods of getting the pAL105 plasmid into our previous transformant. During this process, we also examined other options for our electroporations. It was during this time that we decided to try using E. coli WM54 (a K12 strain with a deletion of the lac operon).

**Results:**
We had no notable results with retrying and tweaking our method for transformation.

# 8/5-8/9

## Trying to transform LasR plasmid into WM54+pAL105 and pAL106 strains

**Who?**
Prajwal, Jason, Jacob

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to transform WM54 + pAL105 and WM54 + pAL106 with LasR Biobrick plasmid. These transformations were performed with positive and negative controls, and the transformants were plated on LB+cam,tet

**Results:**
We were able to obtain transformants after plating, and these plasmids were stably maintained.

# 9/9-9/13

## Fluorescence Assays of LasR part using transformant strains and Spectromax

**Who?**
Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to run 96-well fluorescence assays of three of our strains, WM54 + pAL105, WM54 + pAL105 + LasR, and WM54 + pAL106 + LasR, with the AHLs oxoC12, oxoC8, C4, and C12. These were added in differing concentrations to observe fluorescence and determine the interaction between the LasR-dependent promoter and our system.

**Results:**
We had difficulties given that the concentration of ethyl acetate in our initial control wells appeared to be too high, melting the plate and forcing us to forego the experiment at the time. On 9/14, the plate was run.

# 9/23-9/27

## Fluorescence Assays of LasR part using transformant strains and Spectromax

**Who?**
Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to run 96-well fluorescence assays of four of our strains, WM54 + pAL105, WM54 + pAL105 + LasR, and WM54 + pAL106 + LasR, and WM54 + LasR with the 3 AHLs oxoC12, oxoC8, and C12. These were added in differing concentrations to observe fluorescence and determine the interaction between the LasR-dependent promoter and our system. This was done to obtain triplicates, while also maximizing the controls analyzed while eliminating an unnecessary AHL situation.

**Results:**
A Med Center System Update interfered with the results, so this needed to be redone.

# 9/30-10/5

## Fluorescence Assays of LasR part using transformant strains and Spectromax

**Who?**
Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to run 2 96-well fluorescence assays of four of our strains, WM54 + pAL105, WM54 + pAL105 + LasR, and WM54 + pAL106 + LasR, and WM54 + LasR with the 3 AHLs oxoC12, oxoC8, and C12. These were added in differing concentrations to observe fluorescence and determine the interaction between the LasR-dependent promoter and our system. This was done to obtain triplicates, while also maximizing the controls analyzed while eliminating an unnecessary AHL situation.

**Results:**
We were able to obtain a first and second round of data to work with and compare to others.

# 10/8-10/12

## Fluorescence Assays of LasR part using transformant strains and Spectromax

**Who?**
Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to run 96-well fluorescence assays of four of our strains, WM54 + pAL105, WM54 + pAL105 + LasR, and WM54 + pAL106 + LasR, and WM54 + LasR with the 3 AHLs oxoC12, oxoC8, and C12. These were added in differing concentrations to observe fluorescence and determine the interaction between the LasR-dependent promoter and our system. This was done to obtain triplicates, while also maximizing the controls analyzed while eliminating an unnecessary AHL situation.

**Results:**
The third round of data was obtained for the statistical tests to determine this promoter’s expression.

# 10/14-10/18

## Fluorescence Assays of LasR part using transformant strains and Spectromax

**Who?**
Prajwal

**Protocol:**
Electroporation (Ahmer)

**Notes:**
We attempted to run 96-well fluorescence assays of four of our strains, WM54 + pAL105, WM54 + pAL105 + LasR, and WM54 + pAL106 + LasR, and WM54 + LasR with the 3 AHLs oxoC12, oxoC8, and C12. These were added in differing concentrations to observe fluorescence and determine the interaction between the LasR-dependent promoter and our system. This was done to obtain triplicates, while also maximizing the controls analyzed while eliminating an unnecessary AHL situation.

**Results:**
We were able to obtain a 4th set of plates to gain statistical significance.

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# Team:OhioState/Experiments

[![](https://static.igem.org/mediawiki/2019/7/71/T--OhioState--BlueLogoSmall.png)](https://2019.igem.org/Team%3AOhioState)

* Project

  [Description](https://2019.igem.org/Team%3AOhioState/Description)
  [Design](https://2019.igem.org/Team%3AOhioState/Design)
  [Experiments](https://2019.igem.org/Team%3AOhioState/Experiments)
  [Notebook](https://2019.igem.org/Team%3AOhioState/Notebook)
  [Results](https://2019.igem.org/Team%3AOhioState/Results)
* [Parts](https://2019.igem.org/Team%3AOhioState/Parts)

  [Contribution](https://2019.igem.org/Team%3AOhioState/Contribution)
  [Characterization](https://2019.igem.org/Team%3AOhioState/Measurement)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AOhioState/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3AOhioState/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3AOhioState/Collaborations)
* Judging

  [Judging Form](https://igem.org/2019_Judging_Form?team=OhioState)
  [Safety](https://2019.igem.org/Team%3AOhioState/Safety)
  [Medal Criteria](https://2019.igem.org/Team%3AOhioState/Medal_Criteria)
* Team

  [Team Members](https://2019.igem.org/Team%3AOhioState/Team)
  [Attributions](https://2019.igem.org/Team%3AOhioState/Attributions)

![](https://static.igem.org/mediawiki/2019/8/82/T--OhioState--ExperimentsHero.png)

# Biobrick DNA Extraction

To use the DNA in the Distribution Kit, follow these instructions: ,/p>

*Note: There is an estimated 2-3ng of DNA in each well. When following this protocol, assume that you are transforming with 200-300pg/µL*

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be **red**, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic\* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

# Qiagen Genomic DNA Purification - DNA QIAamp DNA Mini Blood Mini Handbook

Cut up to 25 mg tissue into small pieces, place in a 1.5 mL microcentrifuge tube, and add 180 uL of Buffer ATL.
Add 20 uL of proteinase K, vortex, then incubate at 55 C until the tissue is completely lysed (in water bath, at least 2 hours, can be overnight).

Vortex for 15 seconds, add 200 uL of Buffer AL to the sample, vortex again, incubate at 70 C for 10 minutes.
Add 200 uL of EtOH (96-100%) to the sample, then vortex.

Pipet the mixture from step 4 into DNeasy Mini spin column placed in a 2-mL collection tube. Centrifuge at 8000 rpm for 1 min. Discard flow-through and collection tube.

Place spin column in a new 2-mL collection tube, add 500 uL of Buffer AW1, centrifuge for 1 min @ 8000 rpm. Discard flow-through and collection tube.

Place spin column in 2-mL collection tube, add 500 uL of Buffer AW2, centrifuge for 3 min @ 14000 rpm to dry the membrane. Discard flow-through and collection tube.

Place spin column in clean microcentrifuge tube, pipet 200 uL of Buffer AE directly onto membrane. Incubate at room temp for 1 min, then centrifuge for 1 min @ 8000 rpm.

# Qiagen QIAprep Spin Miniprep Kit

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

# Mach1 OneShot Chemical Transformation Protocol

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube.
9. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

# Addgene Agarose Gel Electrophoresis Protocol

Measure 1 g of agarose.

Double to obtain 2% agarose gel

Add agarose to 100 mL 1x TAE buffer in a microwavable flask.

TBE can substitute for TAE.

Microwave 1-3 minutes until completely dissolved.

Take it out and swirl every 30-45 seconds. Beware surprise boiling. Stop microwaving as soon as boiling occurs; excessive evaporation will change the agarose percentage. Don't let it boil over.

Cool to about 50 °C (about when it is comfortable to keep your hand on the flask), about 5 minutes.

Add ethidium bromide (EtBr) to a final concentration of xxx (usually about 10 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.

Pour slowly into a gel tray with the well comb in place.

Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.

Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified

Add loading buffer to each of your DNA samples.

Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.

Once solidified, place the agarose gel into the gel box (electrophoresis unit).
Fill gel box with 1xTAE (or TBE) until the gel is covered.

Carefully load a molecular weight ladder into the first lane of the gel. Carefully load your samples into the additional wells of the gel.

Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.

Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

# IDT Primer Resuspension Protocol

Briefly centrifuge the tubes before opening them.

Resuspend in dH2O to 100 uM.

Vortex briefly to ensure complete dissolution, then spin with a small tabletop microcentrifuge for a few seconds to pull the liquid back to the bottom of the containers.

Dilute an aliquot with dH2O to 10 uM.

# NEB 2-Step PCR

**Initial Denaturation**

98 C for 30s

**30-35 Cycles**

98 C for 5-10s

72 C for 30-50s/kb

**Final Extension**

72 C for 5-10min

# Biobrick Assembly - Digestion

To assemble an Upstream Part with a Downstream Part into a Destination Plasmid

Digest Upstream Part with EcoRI-HF® and SpeI.

|  |  |
| --- | --- |
| Upstream Part Plasmid: | 500 ng |
| EcoRI-HF: | 1 µl |
| Spel: | 1 µl |
| 10X NEBuffer 2.1: | 5 µl |
| H2O: | to 50 µl |

Digest Downstream Part with XbaI and PstI.

|  |  |
| --- | --- |
| Upstream Part Plasmid: | 500 ng |
| Xbal: | 1 µl |
| Pstl: | 1 µl |
| 10X NEBuffer 2.1: | 5 µl |
| H2O: | to 50 µl |

Digest the Destination Plasmid with EcoRI-HF® and PstI: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdB, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

|  |  |
| --- | --- |
| Destination Plasmid DNA: | 500 ng |
| EcoRI-HF: | 1 µl |
| Pstl: | 1 µl |
| 10X NEBuffer 2.1: | 5 µl |
| H2O: | to 50 µl |

Incubate all three restriction digest reactions at 37°C for 10 (15) minutes and then heat inactivate at 80°C for 20 minutes.

# Biobrick Assembly - Ligation

Ligate the Upstream and Downstream Parts into the digested Destination Plasmid

|  |  |
| --- | --- |
| Upstream Part digestion: | 2 µl |
| Downstream Part digestion: | 2 µl |
| Destination Plasmid digestion: | 2 µl |
| 10X T4 DNA Ligase Buffer: | 2 µl |
| T4 DNA Ligase: | 1 µl |
| H2O: | 11 µl |

Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Transform 2 μl of the ligation product into 50 μl of competent *E. coli* cells (or other suitable host strain). Select using the antibiotic corresponding to the Destination Plasmid.

# Gibson Assembly

Materials:

* gblock (resuspended).
* Linearized vector backbone.
* NEBuilder HiFi DNA Assembly Master Mix.
* H2O.
* Positive control.

Procedure:

1. Use the fragment assembly protocol from the HiFi DNA Assembly table.
2. Each insert must be at least twice the vector. For this reaction – Vector = 0.05 pmol and each Insert = 0.1 pmol. Allowing the total reaction to be under 0.5 pmol.

   Reagent Volume (µl) pSB1C3 backbone: 2.35 BBa\_K2797002: 5.74 Master Mix: 8.09
3. Add the fragments and vector to a PCR tube with the master mix and water if required. 4. Incubate samples on a heat block 60 minutes at 50 °C.

# Electroporation Protocol + Mannitol Glycerol Electrocompetent Wash

\*On the 5430R Centrifuge Maxspeed

1. Take 5 mL of culture and extract 1 mL
2. Spin this down and resuspend with 350 µl chilled water.
3. Insert 250 µl M-G at the bottom to form the bottom layer
4. Spin this down, remove the top and then bottom layer
5. Resuspend in 100 µl M-G solution
6. Add 100-1000 ng DNA
7. Shock and grow in SOC media, plate on LB+cam plates

# **Nitrogen Free Media**

|  |  |
| --- | --- |
| **Ingredients** | **Gms/litre** |
| Sucrose | 20.000 |
| Dipotassium phosphate | 1.000 |
| Magnesium sulphate | 0.500 |
| Sodium chloride | 0.500 |
| Ferrous sulphate | 0.100 |
| Sodium molybdate | 0.005 |
| Calcium carbonate | 2.000 |

**Nitrogen-Deficient Media**

To Nitrogen-Free media add 2mM Glutamate

**Nitrogen Enriched Media**

To Nitrogen-Free media add 100 mM NH4Cl

Autoclave liquid cycle for 1 hour. Some particulate will not go into solution.

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# Team:OhioState/Results

[![](https://static.igem.org/mediawiki/2019/7/71/T--OhioState--BlueLogoSmall.png)](https://2019.igem.org/Team%3AOhioState)

* Project

  [Description](https://2019.igem.org/Team%3AOhioState/Description)
  [Design](https://2019.igem.org/Team%3AOhioState/Design)
  [Experiments](https://2019.igem.org/Team%3AOhioState/Experiments)
  [Notebook](https://2019.igem.org/Team%3AOhioState/Notebook)
  [Results](https://2019.igem.org/Team%3AOhioState/Results)
* [Parts](https://2019.igem.org/Team%3AOhioState/Parts)

  [Contribution](https://2019.igem.org/Team%3AOhioState/Contribution)
  [Characterization](https://2019.igem.org/Team%3AOhioState/Measurement)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AOhioState/Human_Practices)
  [Education and Engagement](https://2019.igem.org/Team%3AOhioState/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3AOhioState/Collaborations)
* Judging

  [Judging Form](https://igem.org/2019_Judging_Form?team=OhioState)
  [Safety](https://2019.igem.org/Team%3AOhioState/Safety)
  [Medal Criteria](https://2019.igem.org/Team%3AOhioState/Medal_Criteria)
* Team

  [Team Members](https://2019.igem.org/Team%3AOhioState/Team)
  [Attributions](https://2019.igem.org/Team%3AOhioState/Attributions)

![](https://static.igem.org/mediawiki/2019/8/80/T--OhioState--ResultsHero.png)

# Nitrogen Fixation

## Goals:

* Isolate the genes necessary for functional nitrogen fixation and assemble them into a single part, rather than the natural form of multiple gene clusters scattered throughout the organism.
* Insert this part into our recipient organism and have it display evidence of nitrogen fixation.
* Examine if our synthetic, nitrogen-fixing organism can actually aid in plant growth (by a significant amount) in model plants.

## How Are We Meeting These Goals:

As detailed in the Notebook section, our initial strategy of focusing on a single temperature for our PCR experimentation did not work out well (only for nif fragments 2 and 3). It was then that we decided to employ a linear gradient scheme to increase the temperature across 12 different plots in the thermocycler (the temperature difference between plots 1 and 2 is the greatest, while the temperature difference between plots 11 and 12 is the smallest). While this turned out to be a successful method for the majority of the fragments, nif fragment 1 still failed to show up on agarose gels. After optimizing and fixing our primers, we were finally able to get nif fragment 1 to show up on a gel.

![](https://static.igem.org/mediawiki/2019/1/10/T--OhioState--anfGel.png)

Anf Cluster (Expected 7220 bp)

![](https://static.igem.org/mediawiki/2019/b/b7/T--OhioState--vnfFrag1Real.png)

vnf Fragment 1(Expected 6683 bp)

![](https://static.igem.org/mediawiki/2019/thumb/0/00/T--OhioState--vnffrag1.png/549px-T--OhioState--vnffrag1.png)

vnf Fragment 2 (expected 5646 bp)

![](https://static.igem.org/mediawiki/2019/thumb/8/82/T--OhioState--nifFrag1.png/556px-T--OhioState--nifFrag1.png)

nif Fragment 1(Expected 10286 bp)

![](https://static.igem.org/mediawiki/2019/2/2e/T--OhioState--nifFrag3.png)

nif Fragments 3 (Expected 3341 bp and 7420 bp)

![](https://static.igem.org/mediawiki/2019/thumb/5/5b/T--OhioState--nifFrag4.png/663px-T--OhioState--nifFrag4.png)

nif Fragment 4 (Expected 9736 bp)

Once we had these PCR products, we performed Gibson assemblies in order to get our desired product (the single part that contains all genes necessary for nitrogen fixation). Once we obtained this product we transformed it into NEB *E. coli* 10 Beta (optimized for large inserts such as ours), and allowed it to grow overnight on LB tetracycline plates. After successful growth, we selected for *E. coli* with the insert on Nitrogen-Free, Nitrogen-Deficient, and Nitrogen-Enriched media under anaerobic conditions with only leucine supplementation. We then attempted to confirm the presence of the insert in colonies isolated from these plates via colony PCR.

![](https://static.igem.org/mediawiki/2019/thumb/b/bb/T--OhioState--bigplate.jpg/935px-T--OhioState--bigplate.jpg)

*E. coli* 10 Beta with Gibson product, growing on tetracycline plate

We are currently selecting for *E.coli* 10 Beta with the correct Gibson product in an anaerobic chamber. If we’re able to see growth in an oxygen-free environment, on nitrogen-free/deficient media, then it will confirm that our recipient organism is in fact able to fix atmospheric nitrogen into bioavailable ammonia (and in enough quantity to support metabolism). We also tested *R.palustris* CGA010 and *P.protegens* PF5 on these media anaerobically

![](https://static.igem.org/mediawiki/2019/thumb/5/5f/T--OhioState--smallplate1.jpg/905px-T--OhioState--smallplate1.jpg)

![](https://static.igem.org/mediawiki/2019/5/5a/T--OhioState--smallplate2.jpg)

![](https://static.igem.org/mediawiki/2019/thumb/c/c6/T--OhioState--smallplate3.jpg/902px-T--OhioState--smallplate3.jpg)

![](https://static.igem.org/mediawiki/2019/4/47/T--OhioState--smallplate4.jpg)

![](https://static.igem.org/mediawiki/2019/thumb/6/68/T--OhioState--smallplate5.jpg/909px-T--OhioState--smallplate5.jpg)

![](https://static.igem.org/mediawiki/2019/thumb/a/ae/T--OhioState--smallplate6.jpg/908px-T--OhioState--smallplate6.jpg)

Note: Media has some inherent particulate. All PF5 plates (- ctrl) showed no growth and all Palustris (+ ctrl) plates showed robust growth.

For future experimentation, we plan to inoculate Arabidopsis thaliana seedlings (our model plant) with our recipient organism, and evaluate whether our organism can supply ammonia to the plant and if it is in high enough levels to support plant growth (instead of just bacterial growth). Additionally, if this organism can support plant growth, we will evaluate whether it does a comparable job to commercial fertilizers.

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IGEM at Ohio State