iGEM 2025 — Team etsu

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## Our Lab Notebooks

#### Note: Lab notes from non-team members are not included in this document

Notebook: CristianaNotebook: ZoeNotebook: ElliotNotebook: JohnNotebook: PCRNotebook: Construct Assembly

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## Soybean promoter molecular cloning

* DESIGN: Each member was tasked with designing primers based on data we gathered through Integrative Genomics Viewer (IGV).
* BUILD: A construct was built through TA Cloning, allowing for digestion of the synthetic construct: the promoter region.
* TEST: Gel electrophoresis was run to verify amplification, cloning, and PCR was successful.
* LEARN: If the primers failed to amplify, we returned to the design phase and repeated until success was achieved.

---

## Assembly of the vector construct

* DESIGN: The vector and each fragment was designed, and the approach of Gibson Assembly and Heat Shock Transformation was suggested for the fragments.
* BUILD: The vector was assembled and amplified within bacteria through PCR, Gibson Assembly, heat shock transformation.
![Promoter](https://static.igem.wiki/teams/5693/images/promoter-part.avif)* TEST: The assembly of the plasmid was confirmed through Colony PCR.
* LEARN: The result of Colony PCR determined whether it was necessary to repeat the cycle for this protocol. A successful result was achieved.

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Identify genes low expression in seeds
![Genes for vitamin expression](https://static.igem.wiki/teams/5693/images/seed-gene-expression.avif)

Finding the promoter sequences from soybean genome:
![Finding promoter sequences](https://static.igem.wiki/teams/5693/images/find-promoter-sequences.avif)

---

### What did we learn? What would we change?

The next cycle would be a bit different for us, as this would be utilized as a template, simplifying the protocol significantly. In a future iteration of the cycle, optimization of the protocol would include following our recorded successful protocol as closely as possible, given its success. We would also design multiple primers at one time in the instance one primer did not work, in order to be effectively prepared and save the time spent redesigning.

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### **Soybean Genomic DNA Extraction**

Purpose:
The purpose of this experiment is to extract genomic DNA from soybean cotyledon and seed tissue to compare the amounts that are able to be extracted. The amounts of DNA for the variable target gene extracted can then be compared for gene expression purposes. To do this, we will prepare samples, lyse cells, extract, and then perform precipitation and washing.

Results:
The extraction of the genomic DNA for the seed was successful. The extraction for the
produced a pellet that could further be purified and tested. This genomic DNA will be used for future experiments when testing the variable promoter region and cloning the
promoter region into a plasmid.

##### Materials:

Equipment:

* Tabletop scale,
* 60 °C water bath,
* tabletop centrifuge,
* 1.5 mL centrifuge tubes,
* blue pestles,
* pipettes (P1000, P200)

Reagents:

* CTAB buffer (2% CTAB, 1% PVP, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA)
* Phenol:Chloroform:Isoamyl alcohol (25:24:1)
* Cold isopropanol
* 70% cold ethanol
* TE Buffer

#### **Protocol**

1. Sample Preparation and Cell Lysis
   * Weigh 200 mg of soybean tissue (cotyledon or seed) using the tabletop scale.
   * Transfer tissue to a labeled 1.5 mL centrifuge tube.
   * Add 500 µL CTAB buffer.
   * Grind tissue with pestle until fully lysed.
   * Incubate at 60 °C for 30 minutes in a water bath.
2. Organic Extraction
   * Centrifuge at 14,000 x g for 5 minutes.
   * Transfer ~200 µL supernatant to a new tube.
   * Add 200 µL PCI (25:24:1); vortex 5 seconds.
   * Centrifuge at 14,000 x g for 1 minute.
   * Transfer 200 µL supernatant to a new tube.
   * Repeat PCI extraction once more to remove residual protein.
3. DNA Precipitation and Washing
   * Add 150 µL cold isopropanol (1:1 ratio) to the sample.
   * Incubate at -20 °C for 30 minutes to precipitate DNA.
   * Centrifuge at 14,000 x g for 10 minutes.
   * Carefully remove isopropanol without disturbing pellet.
   * Add 500 µL cold 70% ethanol to wash pellet.
   * Allow pellet to air dry, then store for further use or resuspend in TE buffer.

### **pENTR-T Plasmid Extraction**

Purpose:The goal of this experiment is to extract a pENT-R plasmid from E. coli that can be used later for gene insertion of the soybean variable target gene. We strive for maximum purity and a high concentration of the plasmid.

Results:The extraction did produce a pENTR-T vector that was consistent with the desired length and could be used for enzyme digestion and insertion of the variable gene.

##### Materials:

Equipment:

* 1.5 mL centrifuge tubes
* Tabletop centrifuge
* DNA spin columns

Reagents:

* E. coli culture (plasmid-bearing)
* Solution I (250 µL): contains RNase A for RNA degradation
* Solution II (250 µL): contains NaOH and SDS for cell lysis
* Solution III (350 µL): neutralization buffer for protein separation
* Wash buffer (500 µL per wash): 70% ethanol in deionized water
* Deionized water (30 µL): for DNA elution

#### **Protocol**

1. Cell Collection
   * Add E. coli culture to 1.5 mL tube in 1 mL increments.
   * Centrifuge at 12,000 × g for 2 minutes; discard supernatant.
   * Repeat 6 times until 6 mL total culture processed and pellet formed.
2. Cell Lysis
   * Add 250 µL Solution I; vortex to fully resuspend pellet.
   * Add 250 µL Solution II; gently invert several times (do not vortex).
   * Mixture becomes clear and viscous (indicating DNA release).
   * Add 350 µL Solution III; invert several times to mix.
   * Centrifuge at 12,000 × g for 10 minutes.
   * Carefully transfer the upper phase (DNA-containing) solution to a DNA spin column.
3. DNA Purification
   * Centrifuge column at 8,000 × g for 2 minutes; discard flow-through.
   * Add 500 µL 70% ethanol wash buffer; centrifuge 8,000 × g for 2 minutes.
   * Discard waste and repeat ethanol wash once more.
   * Perform a dry spin at 12,000 × g for 30 seconds to remove residual ethanol.
4. DNA Elution
   * Place column in a new, labeled centrifuge tube.
   * Add 30 µL deionized water directly to column membrane.
   * Centrifuge at 12,000 × g for 2 minutes.
   * Collect eluted plasmid DNA in tube and store appropriately.

### **Restriction Enzyme Digestion to Make a Vector**

PURPOSE: The goal of this experiment is to digest the plasmid that was extracted previously. This would ensure that the T-DNA was cut from the plasmid and both sections could then be extracted for insertion and transformation.

RESULTS: In some cases, primers were sufficient and vector assembly was successful. In others, primers were redesigned and the process was repeated again with new primers until successful.

#### Reagents and Materials

* 10× NEB 2.1 buffer: 2 µL
* pENTR-T plasmid: 15 µL (10 µL from sample 1, 4 µL sample 2, 1 µL sample 3)
* Deionized water: 2.5 µL
* XcmI restriction enzyme: 0.5 µL (added last)
* 20 µL PCR tube
* Agarose gel components: 1× TAE, agarose, GelGreen, loading dye
* Thermocycler

#### **Protocol**

1. Combine 2 µL buffer + 15 µL plasmid + 2.5 µL DI water in PCR tube.
2. Add 0.5 µL XcmI enzyme last. Remove any enzyme adhering to pipette tip by wiping against tube wall.
3. Incubate in thermocycler at 37 °C for 1 h.
4. Mix digested DNA with 4 µL agarose loading dye, load onto agarose gel.
5. Run gel for 20 min, view to confirm digestion, then run an additional 10 min for clearer band separation.
6. Excise desired DNA fragments with razor and save for gel extraction.

### **Gel extraction of pENTR-T Digestion Product**

PURPOSE: The goal of this experiment is to extract the plasmid DNA fragments and insert the variable gene into the plasmid using previously designed primers.

RESULT: The product of this gel extraction was saved for a later experiment. When it was used for T-A cloning with the promoter region it did show the correct bp length which proves the gel extraction was successful.

#### Materials

Equipment:

* 1.5 mL Eppendorf tubes
* DNA spin columns
* Tabletop centrifuge
* Water bath (set to 55 °C)
* Vortex

Reagents:

* Extraction buffer (450 µL per sample)
* Wash buffer (500 µL per wash)
* Deionized water (15 µL)

#### **Protocol**

1. Gel Dissolution
   * Obtain previously cut and labeled gel slice containing plasmid digestion product.
   * Add 450 µL extraction buffer to the tube using a P1000 pipette.
   * Place tube in 55 °C water bath for ~20 minutes, vortexing periodically to aid dissolution.
   * Ensure gel is fully dissolved before proceeding.
2. DNA Binding
   * Transfer entire dissolved sample to a new, labeled DNA spin column using a P1000 pipette.
   * Centrifuge at 8,000 × g for 2 minutes.
   * Discard flow-through.
3. Washing and Drying
   * Add 500 µL wash buffer to the column.
   * Centrifuge at 8,000 × g for 2 minutes.
   * Discard waste.
   * Perform an additional dry spin at 8,000 × g for 2 minutes to remove residual liquid.
4. DNA Elution
   * Place spin column into a new, labeled 1.5 mL Eppendorf tube.
   * Add 15 µL deionized water directly to column membrane using a P20 pipette.
   * Centrifuge at 12,000 × g for 2 minutes to elute purified plasmid DNA.
   * Cap and label the eluate as pENTR-T, and store for later cloning use.

### **PCR Amplification of the promoter region**

PURPOSE: The goal of this experiment is to successfully clone the variablepromoter in order to insert into the pENTR-T vector. This will confirm that the primers are good and that plant transformation can be performed.

Results in image below. A second amplification was run for those that were not amplified.

![Green test tubes in a row](https://static.igem.wiki/teams/5693/images/enzyme-digestion.webp)

#### Materials

Equipment:

* Micro PCR tubes
* Benchtop thermocycler
* Gel electrophoresis box and viewer
* P20 and P2 pipettes

Reagents:

* 5× GeneScript reaction buffer (10 µL)
* 2.5 mM dNTP mix (5 µL)
* Forward primer (2 µL)
* Reverse primer (2 µL)
* Genomic soybean DNA (2 µL)
* Q5 polymerase (0.5 µL)
* Deionized water (28.5 µL)
* Loading dye (5 µL)
* Agarose gel and DNA ladder

#### **Protocol**

1. PCR Reaction Setup
   * Prepare a 50 µL total reaction volume per sample in micro PCR tubes.
   * Add reagents in the following order:
     1. 28.5 µL deionized water
     2. 10 µL 5× reaction buffer
     3. 5 µL 2.5 mM dNTPs
     4. 2 µL forward primer
     5. 2 µL reverse primer
     6. 2 µL genomic DNA template
     7. 0.5 µL Q5 polymerase
   * Mix gently and briefly centrifuge to collect contents.
2. Touchdown PCR Amplification
   * Load samples into benchtop thermocycler
   * Run the following thermal profile:
     + Initial Denaturation: 98C for 30 seconds
     + Touchdown cycles (x30)
       - Denaturation: 98C for 10 seconds
       - Annealing: gradient 72C -> 50C for 30 seconds
       - Extension: 72C for 1 minute* Store PCR products at -20C until analysis
3. Gel electrophoresis verification
   * Combine PCR product + 5 µL loading dye.
   * Load samples into agarose gel alongside DNA ladder.
   * Run gel for ~10 minutes.
   * Visualize under gel viewer; identify y-TMT promoter band.
   * Cut and save correct-length bands for gel extraction.

### **T-A cloning of Promoter Region**

PURPOSE: The aim of this experiment is to insert the promoter region of the variable gene that had previously been amplified into a digested pENTR-T vector. This will be verified by the length of the PCR product that is 2000bp longer than the original plasmid.

RESULTS: This shows the resulting gel from the TA cloning and ligation. The band lengths for all samples are consistent with the desired length of the ligated plasmid.

![Green test tubes](https://static.igem.wiki/teams/5693/images/ta-cloning.webp)

#### Materials

Equipment:

* PCR tubes
* Thermocycler
* Vortex

Reagents:

* Extracted promoter DNA (from gel extraction)
* 10× PCR buffer (1.5 µL)
* dATP (1 µL)
* Taq polymerase (0.25 µL)
* Digested pENTR-T vector (2 µL)
* T4 DNA ligase buffer (1.5 µL)
* T4 DNA ligase (1 µL)
* ‘A’ reaction product (10.5 µL)
* Loading dye (5 µL)

#### **Protocol**

1. 'A' Reaction - Addition of A-Tails
   * Begin with 15 µL of gel-extracted DNA, using 12.25 µL for the reaction.
   * In a PCR tube, add reagents in the following order:
     1. 12.25 µL promoter DNA
     2. 1.5 µL 10× PCR buffer
     3. 1 µL dATP
     4. 0.25 µL Taq polymerase (added last)
   * Incubate at 72 °C for 30 minutes to add adenine (A) overhangs to both DNA ends.
3. Concentration check
   * Mix 2 µL of A-reaction product with 2 µL loading dye.
   * Run on agarose gel for 10 minutes to confirm DNA integrity and concentration.
4. Ligation Reaction
   * In a PCR tube, combine the following:
     1. 2 µL digested pENTR-T vector
     2. 1.5 µL T4 DNA ligase buffer
     3. 10.5 µL A-tailed promoter DNA
     4. 1 µL T4 DNA ligase (added last)
   * Mix gently and incubate at room temperature for 3 hours to ligate insert into vector.
5. Verification and Storage
   * Add 5 µL loading dye to ligation mix.
   * Run ligation product on agarose gel to confirm insertion (expect 2000 bp size increase).
   * Store successfully ligated plasmids at -4 °C for future transformation.

To make 10 µM working solutions, 10 µL of the 100 µM stock was diluted with 90 µL DI water in a labeled microtube indicating primer name, direction (forward or reverse), and concentration.

### **Q5 PCR**

PURPOSE: The purpose of Q5 PCR is to assemble the plasmid and fragments, assembling pCNHP with the PSYpro and pCNHP with RUBY.

RESULT: After a few trial rounds of PCR, the ideal conditions for PCR were found, successfully assembling the product.

Two rounds of PCR were performed: one to unite pCNHP and PSYpro, and another to unite pCNHP and RUBY

Q5 Reaction Mix:

* 30 µL DI water
* 10 µL 5× NEB Reaction Buffer
* 5 µL 2.5 mM dNTP
* 2 µL Forward Primer
* 2 µL Reverse Primer
* 0.5 µL Q5 Taq Polymerase

95°C for 2 min → [95°C for 30 s, 68°C (–0.5°C per cycle) for 30 s, 72°C for 1.5 min] × 37 cycles → 72°C for 2 min

### **Gel Extraction**

PURPOSE: The gel extraction method was used to isolate the newly assembled PCR product in its purest form.

RESULTS: 20 microliters of concentrated and pure product were produced from the extraction.

#### Reagents and Materials

* 1 g agarose + 50 mL TAE buffer (2% gel)
* 12 µL loading dye
* Extraction buffer (450 µL)
* Wash buffer (2 × 500 µL)
* Deionized water (20 µL for elution)
* DNA spin column + 1.5 mL Eppendorf tubes

#### **Protocol**

1. Run 2% agarose gel with PCR product + 12 µL dye until ~⅔ of gel is completed.
2. Excise target band and place in 1.5 mL tube.
3. Add 450 µL extraction buffer, incubate at 55 °C until dissolved.
4. Transfer to spin column, centrifuge 8000 × g / 2 min, discard waste.
5. Add 500 µL wash buffer, centrifuge 8000 × g / 2 min, discard waste.
6. Repeat wash once more under same conditions.
7. Centrifuge empty column 12000 × g / 30 s to dry.
8. Place in new 1.5 mL tube, add 20 µL DI water, centrifuge 12000 × g / 2 min for elution.
9. Store eluted DNA at −20 °C.

### **DNA Concentraion Verification by Agarose Gel**

PURPOSE: After gel extraction, the concentration of each product should be checked through gel electrophoresis.

RESULT: A prominent band was observed for each at approximately the corresponding bp, indicating high concentration.

![PCR bands](https://static.igem.wiki/teams/5693/images/concentration-verif.webp)

Order: RUBY (approx 1kb), PSY (approx 500pb), pCNHP (approx 1.2kb)

#### Reagents and Materials

* Agarose: 0.7 g
* TAE buffer: 50 mL (1.5% gel)
* Loading dye: 2 µL
* DNA sample: 2 µL

#### **Protocol**

1. Prepare 1.5% agarose gel (0.7 g in 50 mL TAE).
2. Mix 2 µL DNA sample + 2 µL loading dye, load into gel.
3. Gel was run at 70 volts for 30 minutes.

### **Gibson Assembly**

PURPOSE: Gibson Assembly is a method of assembling multiple DNA fragments without the use of a restriction enzyme. This was used for assembling PSYpro and RUBY.

RESULTS: Assembly of the fragments was successful.

#### Reagents and Materials

* PSYpro gel extraction DNA: 2 µL
* RUBY gel extraction DNA: 2 µL
* pCNHP plasmid: 1 µL
* 2× Gibson Assembly Mix: 5 µL
* Thermocycler

#### **Protocol**

1. Combine 2 µL PSYpro + 2 µL RUBY + 1 µL pCNHP + 5 µL 2× Gibson Mix in a reaction tube.
2. Incubate in thermocycler at 50 °C for 1 hour (Incubate mode).

### **LB Agar Plate Preparation with Kanamycin**

PURPOSE: These plates offer an antibiotic selection that our plasmid, pCNHP, has a resistance to, eliminating any non-resistant colonies and allowing our plasmid to thrive. They are the medium the bacterial will grow upon and where they will derive their nutrients from.

RESULTS: No contamination was observed.

#### Reagents and Materials

* LB agar: 4 g
* DI water: 100 mL
* Antibiotic: Kanamycin
* Petri plates

#### **Protocol**

1. Dissolve 4 g LB agar in 100 mL DI water.
2. Autoclave to sterilize.
3. Allow medium to cool, then add Kanamycin.
4. Pour 20 µL per plate once cooled.
5. Let solidify and store plates until use

### **Transformation of Competent Cells with Gibson Assembly Product**

PURPOSE: Heat shock transformation was used to insert our plasmid into competent E.coli, allowing it to replicate and thrive before using the Kanamycin+ plates to weed out the plasmids that are not our assembled one.

RESULTS: Heat shock was successful, as colonies were observed.

![Agar plates with bacteria colonies](https://static.igem.wiki/teams/5693/images/plates.webp)

#### Reagents and Materials

* Heat-shock competent cells (C2987)
* Gibson assembly product: 10 µL
* SOC medium: 100 µL
* LB agar plates with Kanamycin
* Ice, water bath, incubator

#### **Protocol**

1. Add 10 µL Gibson assembly product to competent cells and keep on ice for 30 min.
2. Heat shock in 42 °C water bath for 1 min, then return to ice for 2 min.
3. Add 100 µL SOC medium, incubate at 37 °C for 30 min.
4. Spread cells onto LB agar + Kanamycin plates.
5. Incubate at 37 °C overnight.

### **Colony PCR and Gel Verification of Transformants**

PURPOSE: Colony PCR is used to determine if a colony from the aforementioned E. Coli plate contains the plasmid carrying the target gene(s). PCR is used to amplify the insert fragment if it is present, which shows up as a band on gel electrophoresis. If there is no insert present, it will not be amplified, and no band will be shown on the gel electrophoresis. A positive control of the insert itself is run to compare and verify the band is not non-specific.

RESULTS: A band was observed that aligned with that of the positive control.

![PCR Bands](https://static.igem.wiki/teams/5693/images/pcr-1.webp)
![PCR Bands](https://static.igem.wiki/teams/5693/images/pcr-2.webp)

#### Reagents and Materials

* LB broth with Kanamycin: 5 PCR tubes (~5 mL each)
* Transformed C2987 colonies
* PCR mastermix:
  + 10 µL 10× buffer
  + 10 µL dNTP
  + 71.5 µL H₂O
  + 1.25 µL CNHP-PSY-F primer
  + 1.25 µL PSY-RUBY-R primer
  + 1 µL Taq polymerase
* Agarose gel: 0.7 g agarose in 50 mL TAE (1.5%)
* Loading dye: 4 µL 5× for samples, 2 µL 5× for positive control
* Thermocycler

#### **Protocol**

1. Pick a single colony from transformed C2987 cells, inoculate one LB + Kan tube, mix, and repeat for 5 tubes, labeling serially. Incubate at 37 °C for 1 h.
2. Prepare PCR mastermix (see reagent volumes above).
3. Aliquot per tube:
   * 2 µL 10× buffer
   * 2 µL dNTP
   * 14.3 µL DI water
   * 0.25 µL CNHP-PSY-F primer
   * 0.25 µL PSY-RUBY-R primer
   * 0.2 µL Taq polymerase
   * 1 µL incubated colony culture
4. Run thermocycler:
   * 95 °C 2 min
   * 36 cycles: 95 °C 30 s, 56 °C 30 s, 72 °C 30 s
   * Final extension: 72 °C 2 min
   * Hold: 22 °C 2 min, lid 100 °C
5. Load full PCR product on 1.5% agarose gel with:
   * 4 µL 5× dye per sample
   * Positive control: 5 µL DNA + 2 µL 5× dye
6. Run gel at 70 voltz for 40 minutes.

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## Our Results

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1. 1. The promoters from those genes in soybean are cloned for further mutagenesis and Arabidopsis transformation.
   ![Vitamins](https://static.igem.wiki/teams/5693/images/vitamins-genes.avif)
2. 2. Performance of the trained CNN model (~80% accuracy measured by AUROC). With this model, we can predict whether a mutated promoter can be expressed in seed or not.
   ![AUROC map](https://static.igem.wiki/teams/5693/images/auroc.avif)
3. 3. Tested non-seed promoter expression in *Nicotiana tabacum*
   ![Tobacco plant](https://static.igem.wiki/teams/5693/images/tobacco.avif)

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