iGEM 2021 — Team NDSU

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# Team:NDSU/Notebook

# Notebook

Below is our team lab notebook detailing our work each day throughout the project.

### 10/19/21

Mia Haugan, Erica Crawford: Measured the distance the fungus had grown over the colony, starting at the edge of the colony closest to the fungal plug. Measured in mm. This was after 6 days of growth. R. solani sugarbeet had completely overgrown the colonies and couldn’t be recorded.

### 10/13/21

Jake Schumacher: "I stocked GG6a, GG8c, GG9a, GG9c, GG10a in the –80C freezer. Added 5 drops of 80% glycerin to 900uL of each culture in a cryovial, then placed them in the freezer. "

Mia Haugan, Jake Schumacher: Started fungal inhibition assay of using 2A, 3A, 3B, 4B, 4D, and RFP. 3 replicates on PDA plates both with and without Km, and using F. oxysporum 10-1, F. oxysporum 10-5, R. solani Agassiz, and R. solani sugarbeet. This made for a total of 24 plates. Using protocol from 10/7 from first fungal assay. Then placed in 28C incubator.

### 10/12/21

Jake Schumacher: "I Started liquid cultures of GG2A, GG3A, GG4B, GG4D, GG5A, GG6A, GG8C, GG9C, and GG10A. The test tubes were incubated in a 37C shaker overnight."

### 10/9/21

Jake Schumacher: I plasmid Prepped GG6a, GG8c, GG9a, GG9c, GG10a from the liquid cultures started on 10/7 (they were pulled out of the incubator and placed in a 9C refrigerator on 10/8) using the Qiagen Miniprep kit and following its protocol.

### 10/7/21

Drew Jordahl: Struck successful (verified by 10/6/21 PCR) GG clonings onto LBA+Km plates and with the same loop inoculated LB+Km broths. Incubated the plates overnight at 37C and the test tubes in a 37C shaker overnight. Specifically, GG6a, GG8c, GG9a, GG9c, GG10a. GG9 plate was a little odd, there was this mysterious brownish tint to 3 of the 4 quadrants, GG9c was the only one without the brown tint.

Drew Jordahl: Counted the colonies on the GG6-10 GG cloning transformation plates:

GG6 10-1 plate: 153 colonies

GG7 10-0 plate: 8 colonies, 10-1 plate: 1 colony

GG8 10-1 plate: 74 colonies

GG9 10-2 plate: 247 colonies

GG10 10-1 plate: 402 colonies

Mia Haugan: Fungal inhibition assay for 2A, 3A, 3B, 4A, 4D, and RFP (control). Did 4 plates, F. oxysporum 10-1, F. oxysporum 10-5, R. solani from sugarbeets, and R. solani agassiz. Placed in the 28C incubator.

Mia Haugan: Assay was done by spotting 10ul each of the samples (already normalized to OD 1) on a plate, approx. 2.75cm away from the center and equidistant from the other samples. Then a fungal plug was placed in the center.

### 10/6/21

Jake Schumacher: Started broth cultures of 2A, 3A, 3B, 4A, 4D, and RFP. This is for the fungal assay tomorrow

Jake Schumacher: PCR Screening of GGR3-10

![](https://static.igem.org/mediawiki/2021/5/5a/T--NDSU--OctoberSixthGel.jpg)

Order from left to right (Top): GeneRuler 1kb DNA Ladder, -, GGR3a, GGR3b, GGR4a, GGR4b, GGR6a, GGR6b, -, Ladder, -, GGR6c, GGR6d, GGR7a, GGR7b, GGR7d, -, -, -, Ladder
Order from left to right (Bottom): Ladder, GGR8a, GGR8b, GGR8c, GGR8d, GGR9a, GGR9b, -, Ladder, -, GGR9c, GGR9d, GGR10a, GGR10b, GGR10c, GGR10d, -, -, Ladder
Expected amplicons: GGR3 = 1581, GGR4 = 2732, GGR6 = 1674, GGR7 = 804, GGR8 = 3260, GGR9 = 1548, GGR10 = 1764
All bands look to be expected sizes except: GGR4a, all three GGR7, GGR10d.

### 9/29/21

\*Update\*

### 9/15/21

Zach Tiedeman: Measured the concentration of the gene blocks and DVK\_EF with the nanodrop.
![](https://static.igem.org/mediawiki/2021/8/85/T--NDSU--SeptFifteenthChart.jpg)
GG 6 & 7 created using golden gate cloning protocol.

### 9/14/21

![](https://static.igem.org/mediawiki/2021/a/ad/T--NDSU--SeptFourteenthGel.jpg)

\*Update\*

### 9/8/21

Zach Tiedeman, Adele Malone, Everett Glower: Did a digest, but used the wrong enzyme (Bpi1/Bbs1 is needed, NOT BsaI!!!). Lost work. We have a gel and a ladder/die mix in the fridge for the next try – we figured out our mistake before putting the gel in TAE buffer.

### 9/2/21

Madilyn Smith, Everett Glower, Zach Tiedeman: Did QIAprep plasmid miniprep of GG2(A-D), GG3(A-D), GG4(A-D), and GG5. We placed the plasmids in the –20C freezer box, and updated the table under “8/16”.

Madilyn Smith, Everett Glower, Zach Tiedeman: The caps for the last centrifuge cycle broke during the rotations. Keep caps pointing in a clockwise direction to prevent damage from torque in future centrifugations.

### 9/1/21

Zach Tiedeman: I transferred each of the colonies that I streaked from 8/31 into 3 mL of LBB+KB broth, and placed them in the incubating shaker overnight, along with a control. The vials are labeled according to their strain (GG2-5) and their position on the 4-colony streak plate (A-D) Two notes:

-GG2 colony “C” (as labeled on the streak plate) did not exist. I suspect that I missed streaking it on 8/31. In an attempt to have better odds of getting a successful product, I instead created TWO vials from colony “D”, with the second one labeled GG2D2

-GG5 was the RFP-producing colony. Because we already have a viable plasma from this strain (and the colonies show visible signs of production), I only transferred one as the control for the plasma extraction, digest, and electrophoresis run.

### 8/31/21

Zach Tiedeman: transferred 4 colonies from each of the plates in the previous step to stock plates. I used one plate for each strain, dividing the plate into 4 quadrants, and labeling them A, B, C, and D. To do tomorrow: Transfer each of those colonies into 3.5 mL of LBB + KM broth.

### 8/26/21

Zach Tiedeman: transformed competent E. coli with GG#2-5. (see Competent E. coli transfer in protocol list), using 2 uL each of GG#2-5.

Zach Tiedeman, Caroline Osborne, and Everett Glower: Dilution protocol with GG #2-5.

### 8/25/21

\*All work was lost for the day because the UV light was left on in the hood with all of the samples

Zach Tiedeman: transformed competent E. coli with GG#2-5. (see Competent E. coli transfer in protocol list), using 2 uL each of GG#2-5.

Zach Tiedeman, Caroline Osborne, and Erica Crawford: Dilution protocol with GG #2-5.

### 8/24/21

Jake Schumacher: 30uL of DNA blocks (10ng/uL) were distributed to 3 separate centrifuge tubes for all 3 of the newly created DNA block stocks (ChiA, ChiB, rhlAB).

Zach Tiedeman & Taylor Pennington: We added 100 uL of millipore H2O to the ChiA, ChiB, and rhlAB DNA blocks. We vortexed them for 1~2 seconds, twice, then spun them down.

Zach Tiedeman & Taylor Pennington: We assembled and ran a golden gate cloning procedure for ChiA (GG#2),ChiB (GG#3), rhlAB (GG#4), and RFP (GG#5), using plasmids #2, #6, #8, and #13. We used the Golden Gate Protocol, with specifics as listed below. Two items of note: we knocked over the PCR tube for GG#4, with no visible spillage, and we accidentally used a 10 uL pipette for a 1.2 uL distribution of plasmid #13. We ran in the thermocycler under “Jake - BsaI GG”

GG#2-4 all used: 1.0 uL BsaI GG, 2.0 uL T4 ligase, 1.6 uLplasmid #2, 2.0 uLplasmid #6, 1.1 uL plasmid #8, 1.2 uLplasmid #13

GG#2 used: 5.2 uL ChiA, 5.9 uL millipore H2O

GG#3 used: 2.9 uL ChiB, 8.2 uL millipore H2O

GG#4 used: 5.9 uL rhlAB, 5.2 uL millipore H2O

GG#5 used: 1.1 uL plasmid #7 (RFP sequence), 10.0 uL millipore H2O

### 8/23/21

Zach Tiedeman: Ran first golden gate cloning! Components used: 1 uL BSA1GG, 2 uL T4 DNA ligase buffer, 1.6 uL #2, 2.0 uL #6, 1.1uL #7, 1.1uL #8, 1.2uL #13, 10.0 uL Millipore H2O. Started with H2O, then T4, #2, #6, #7, #8, #13, and finally BSA1GG. Ran in thermocycler under “Jake - BsaI GG”.

ZPT: Measured the concentration of the components for the first round of golden gate cloning using the nanodrop. Results:
![](https://static.igem.org/mediawiki/2021/f/fa/T--NDSU--AugTwentyThreeChart.jpg)

Zach Tiedeman: The 260/230 ratios on both #6 and # 8 are concerning. We are planning to move forward with Golden Gate cloning, and revisit these two if our target plasmid fails to assemble.

Caroline Osborne: GG#1 competent E. coli transformation (see protocol list), used 2 uL

Zach Tiedeman & Adele Malone: Spread Plated GG #1 Transformation after diluting (see protocol list) and placed in 37C degree incubator until tomorrow.

Zach Tiedeman & Adele Malone: Poured LBA+Km plates and stored them in the 8C refrigerator.

### 8/20/21

Zach Tiedeman, Marissa Meyer, and Everett Glower: We ran a plasmid prep on DVA\_AG, DVK\_AF, DVK\_EF, and DVK\_AE. We used our modified QIAprep Spin Miniprep Kit protocol (find on the top of the inside of our miniprep kit (250) box. Modifications are marked on the protocol). We used 2 mL for each.

\*Update - Digestion of plasmids\*

Zach Tiedeman and Marissa Meyer: Marissa and I ran the gel. Marissa prepped the gel. The following recipes were used for the samples loaded into the gel: 25 uL plasmid digest solution + 5 uL dye, and 15 uL GeneRuler 1kb DNA Ladder + 3 uL dye

Zach Tiedeman: Gel Electrophoresis of BsaI digest
![](https://static.igem.org/mediawiki/2021/9/93/T--NDSU--AugTwentyGel.jpg)

Jake Schumacher: Order from left to right: GeneRuler 1kb DNA Ladder, “12 DVA\_AG”, “14 DVK\_AF”, “15 DVK\_EF”, “13 DVK\_AE”.
Bsa1 digest of the 4 plasmids prepped on 8/20 because of their inconclusive verification on 8/19. We expected a backbone fragment at 2108bp for the AmpR #12 plasmid and a 2235bp backbone fragment for the 3 KmR plasmids (13-15). All 4 digests should have a 506bp fragment too (feint but looks like “14 DVK\_AF” still has it). “12 DVA\_AG” and “14 DVK\_AF” both show their expected fragment sizes. 15 DVK\_EF” and “13 DVK\_AE” have a weirdly big backbone, but they both have the 506bp fragment. Conclusion: Trust: “12 DVA\_AG” and “14 DVK\_AF.” Sketchy, but might still work: “15 DVK\_EF” and “13 DVK\_AE.”

### 8/19/21

Zach Tiedeman: distributed 4 tubes of KAN + LBB @ 10 mL/tube. Distributed 2 tubes of AMP + LBB @ 10mL/tube. Transferred DVK\_AF, DVK\_EF, and DVK AE to one KAN tube each. Transferred DVA\_AG to a AMP tube. Kept one KAN and one AMP tube for a negative controll. Placed all tubes in incubation room on shaking platform.

Zach Tiedeman: mixed 80 mL of KAN + LBB, using 5uL of KAN per 10 mL of LBB.

40 uL KAN

80 uL LBB

Mixed on a bench, using flame to sterilize. Did not like – will use a hood in the future.

Jake Schumacher: Digested all 15 plasmid preps from 8/18 with Bsa1 restriction enzyme (Reaction mix: 5uL Plasmid + 0.5uL Bsa1 + 2.5uL CutSmart Buffer + 17uL MiliPore Water). 20 minutes at 37C and 10 minutes at 80C.) 5uL of “EZ Vision® DNA Dye as Loading Buffer, 6X” was added to the 25uL of digested plasmid. Then the digestion reactions were then loaded into a 1.5% agarose gel and run for 60 minutes at 100V next to “GeneRuler 1kb DNA” ladders.

![](https://static.igem.org/mediawiki/2021/7/74/T--NDSU--aug_19_image.png)

Order: GeneRuler 1kb ladder on the ends. The top is plasmids 1,2,3,4,5,6,7,8 from left to right and the bottom is 9,10,11,12,13,14,15 from left to right.

Bsa1 digest of all 15 plasmids prepped on 8/18. Expected backbone fragment at 2108bp for all the AmpR plasmids (1-12) and a 2235bp backbone fragment for the 3 KmR plasmids (13-15). Most of the plasmids have smaller fragments that were too small to show up, but “7 E1010m(RFP)\_CD” shows it’s 689bp fragment and “11 DVA\_AF” and “12 DVA\_AG” show their 506bp fragments. Starts to get hairy between 12-15 though.

Conclusion:

Trust: 1-11

Questionable: 12-15. 12(looks correct except for that really big/high band), 13(looks like there is the correct 2235bp band and then one a bit higher… don’t actually know the true digestion pattern of 13 or 14 because Addgene doesn’t have a full sequence, but in theory should look identical to 15), 15(looks like the backbone is ~3300bp instead of 2235bp, but you can see the 506bp fragment...).

Wrong: 14 (or at least to feint to tell)

### 8/18/21

Jake Schumacher: (Mia and Caroline) Each of the 15 strains was plasmid prepped using 1mL of overnight liquid culture using the Qiagen Miniprep kit and following its protocol. Each strain was additionally stocked into the –80C Freezer in our own box (900uL of overnight liquid culture + 6 drops of 80% glycerol).

### 8/17/21

Mia Haugen: Liquid cultures were done, in duplicate, for all of the E. coli. Then they were placed in the 37C room in the shaker overnight

### 8/16/21

Mia Haugen: The E. coli containing the necessary genetic components were struck onto antibiotic LB plates and placed in the 37C incubator.

![](https://static.igem.org/mediawiki/2021/8/8f/T--NDSU--aug_16_image_table.png)

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# Team:NDSU/Engineering

### ★ ALERT!

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Delete this box in order to be evaluated for this medal criterion and/or award. See more information at  [Instructions for Pages for awards](https://2021.igem.org/Judging/Pages_for_Awards).

# Engineering Success

### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

Please see the [2021 Medals Page](https://2021.igem.org/Judging/Medals) for more information.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

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# Team:NDSU/Experiments

# Experiments

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

# Cloning experiments

Our experiments were based on a multi step process, which can be best summarized by:

* 1. Growth of parts from a CIDAR MoClo parts kit ordered from add gene.
* 2. Plasmid extraction.
* 3. Restriction Digest & part verification.
* 4. Golden Gate cloning.
* 5. Transformation.
* 6. Culture.
* 7. Plasmid extraction.
* 8. Restriction Digest & completion verification.

Finally, after we completed this cycle for 3 separate antifungals, we set up an assey to test their efficacy against *Rhizoctonia solani.*

### Golden gate cloning

Golden gate cloning was performed using the NEB® Golden Gate Assembly Kit with a CIDAR MoClo parts kit from the add gene repository, and a thermocycler. Our protocol for each cloning run was based on the NEB Golden Gate Assembly Kit QuickStart protocol for the 25 ul recipe, with two exceptions. First: we utilized a plasmid backbone from our parts kit instead of the generic one from the protocol. Second: we entered the DNA length of each of our parts and the concentration of our plasmid extract into a calculator, and used the correct amount of plasmid solution to have 40 f Mol of each of our parts in our solution.

### Restriction digest

* 5uL plasmids
* 0.5uL restriction enzyme
* 2.5uL CutSmartBuffer
* 17uL Millipore ultrapure water
* Allow reaction to run for 20 minutes at varying temperatures based on the restriction enzyme used

### Plasmid prep

Our plasmid extractions were perfomed using a QIAprep miniprep kit. We used the centrifuge protocol from Qiagen Miniprep kit.

### Transformation

Used the NEB 5-alpha competent *E. coli* (high efficiency) protocol

### Screening process

* Transfer 4 transformed *E. coli* colonies to new antibiotic plate
* Plasmid prep selected colonies
* Run the prepped plasmids on an electrophoresis gel and verify plasmid

# Fungal Assay

This experiment was done to observe any possible antifungal effect from the transformed *E. coli* on the different fungal variants

* Grow verified *E. coli* colonies overnight in Luria-bertani broth plus kanamycin

### What should this page contain?

* Protocols
* Experiments
* Other cool stuff about FUNGUS

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NDSU/Results

# Results

Below are the results of our major experiments. While we did not have greatly significant success with our *E.coli* inhibiting *R.solani*, we hope to continue to adjust our design and see if can achieve better results.

### Fungal Assay Results

For our fungal assay, we measured the distance of overlap of the fungal growth over the *Escherichia coli* colonies. There was very little visible inhibition, and the fungus was found growing over most of the colonies. To the right is a table containing the measurements. There are 3 sets of 6 plates. A fourth set of plates were prepared, however, these were completely overgrown and not measurable.

##### What does this mean?

Our results did not indicate any significant inhibition of *Rhizoctonia solani* by the *Escherichia coli* with the Chitinase A, Chitinase B, or rhamnolipid genes. While we succeeded in inserting this gene, our results suggest that we failed to express it, or express it effectively.

##### Moving Forward

Though we failed to express antifungals in our *E.coli* this time around, we can once again attempt to express then by experimenting with expression regulation, using different antifungal genes, adding sequences that would allow for secretion, or by breaking open the cells to possibly release the antifungals.

***An example plate from our fungal assay:***

![](https://static.igem.org/mediawiki/2021/8/89/T--NDSU--Fungal_Assay_Plate_2_Ag2.jpg)

![](https://static.igem.org/mediawiki/2021/d/d7/T--NDSU--Fungal_Assay_Results_2.png)

* RFP: Red Fluorescent Protein
* 2A: Chitinase A
* 3A: Chininase B selection 1
* 3B: Chitinase B selection 2
* 4B: Rhamnolipid selection 1
* 4D: Rhamnolipid selection 2

### Project Achievements

This season, we had some successes and some roadblocks yet to overcome.

Successful procedures:

* Insertion of Chi A, Chi B, and Rhamnolipid genes into seperate expression vectors, which were successfully transformed into *Escherichia coli*.
* Completion of a fungal assay of three strains of *Rhozoctonia solani* against 6 different *Escherichia coli* growths, one of which was our negative control (RFP) (inhibition not very successful).

Unsuccessful procedures:

* Expression of the antifungals to a point that significantly inhibits *Rhizoctonia solani* on PDA and PDA+Km