iGEM 2018 — Team UNebraska-Lincoln

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# Team:UNebraska-Lincoln/Notebook

# UNL 2018 Improving Early Detection of The Emerald Ash Borer

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[Description](https://2018.igem.org/Team%3AUNebraska-Lincoln/Description)
[Design](https://2018.igem.org/Team%3AUNebraska-Lincoln/Design)
[Experiments](https://2018.igem.org/Team%3AUNebraska-Lincoln/Experiments)
[Notebook](https://2018.igem.org/Team%3AUNebraska-Lincoln/Notebook)
[InterLab](https://2018.igem.org/Team%3AUNebraska-Lincoln/InterLab)
[Contribution](https://2018.igem.org/Team%3AUNebraska-Lincoln/Contribution)
[Model](https://2018.igem.org/Team%3AUNebraska-Lincoln/Model)
[Results](https://2018.igem.org/Team%3AUNebraska-Lincoln/Results)
[Attributions](https://2018.igem.org/Team%3AUNebraska-Lincoln/Attributions)

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[Basic Parts](https://2018.igem.org/Team%3AUNebraska-Lincoln/Basic_Part)

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[Human Practices](https://2018.igem.org/Team%3AUNebraska-Lincoln/HumanLanding)

[Silver HP](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Silver)
[Integrated and Gold](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Gold_Integrated)

# Notebook

---

---

## **Detailed protocols can be found on our experiments page.**

## **Complete notebook can be found on this page: [NOTEBOOK](https://2018.igem.org/Team%3AUNebraska-Lincoln/Notebookdaytoday)**

### **Week 1 – June 4th-8th:**

New team members went through a two-week training program. The team learned how to make LB Broth and agar plates. Training was done on how to prepare chemically competent cells and the respective transformation. PCR for mCherry gene was performed and then analyzed using gel electrophoresis.

### **Week 2 – June 11th-15th**

Training was continued till the end of the week. DNA recovery was performed on the PCR products. Training was done on molecular cloning techniques SLIC and Subcloing.

### **Week 3 – June 18th-22nd**

The third week consisted mainly of InterLab calibrations. We received and streaked DH5-α E. coli hosting plasmids 100167, 100168, and 100169 separately. These plasmids were purchased from Addgene and are the basis of our experiments.

### **Week 4 – June 25th-29th**

The bulk of the InterLab study was conducted by Rachel and Drew. Initial minipreps and transformations into GeneHog E. coli were performed by Gabe.

### **Week 5 – July 2nd-6th**

Two fragment-Sequence and Ligation Independent Cloning (SLIC) was led by Gabe and advised by Jarek. The first attempt was unsuccessful. Second attempt was initiated. Glycerol stocks of the original order were made by Rachel.

### **Week 6 – July 9th-13th**

The team prepares protocol for sesquiterpene assay(s) and starts preliminary in vivo experiment on the originally ordered plasmids, 100167 and 100169. Second SLIC attempt failed due to biological contamination. (Link to worst gels). Third SLIC attempt completed and sent for sequencing.

### **Week 7 – July 16th-20th**

Initial sequencing looks good. More primers are ordered to sequence middle of inserted gene. With the help of DJ, Nic and the team prepared GC-MS samples from last week’s experiment. The goal was to detect amorphadiene, but it did not show on the resulting chromatograms. Restriction digest and PCR was used to help verify a successful cloning. Glycerol stock was made of the double plasmid transformation (100167 and 100169-TPS4).

### **Week 8 – July 23rd-27th**

The sequencing of the cloned plasmid was confirmed. A new assay was carried out, led by Rachel. Three cultures were used: 2 plasmid system with pADS with IPTG induction, 2 plasmid system with pTPS4 with IPTG induction, and a GeneHog control. Nic performed ligation cloning for the codon optimized variant of TPS4. Transformation looks successful.

### **Week 9 – July 30th-August 3rd**

PCR was used on the most recent clone. The PCR product was sent in for sequencing. Rachel prepared GC-MS samples and analyzed the results. The samples from the ADS cultures showed presence of amorphadiene. The samples from the pTPS4 cultures were inconclusive. A glycerol stock of a pTPS4-CO and 100167 double transformation was made by Rachel. The sequencing for the pTPS4-CO plasmid was later confirmed.

### **Week 10 – August 6th-10th**

Drew made first batch of Terrific Broth. Terrific Broth will be used for the rest of the assays. Our third assay (reported in results as assay 1 since it was the first assay with pTPS4-CO) was carried out with three different cultures: 2 plasmid system with pADS, 2 plasmid system with pTPS4, and 2 plasmid system with pTPS4-CO. Cell pellets and the remaining octane layer were saved.

### **Week 11 – August 13th-17th**

GC-MS samples were prepared and run by Rachel and Howard. This week Drew attempted SDS-PAGE. All attempts failed. Future work will look into protein expression.

### **Week 12 – August 20th-24th**

Our fourth assay (reported in results as assay 2) was ran this week led by Rachel and Nic. Cell pellets and the remaining octane layer was saved. Classes begin at UNL.

### **Week 13 – August 27th-31st**

Another assay, the fifth (known as assay 3), was ran this week. Caryophyllene started to be used as an internal standard in GC-MS analysis to quantify sesquiterpene production.

### **Week 14 – September 3rd-7th**

Re-analysis of previous assay samples using the internal standard failed. Made and tested a different internal standard concentration. Re-analyzed two sample sets with new internal standard.

### **Week 15 – September 10th-14th**

This week and every week after, Rachel has worked on GC-MS sample prep and analysis.

### **Week 16 – September 17th-21st**

Nic begins work to clone the TPS4-CO coding sequence into the iGEM submission vector, pSB1C3. Initial ligation for cloning failed.

### **Week 17 – September 24th-28th**

Sixth and final assay (reported as assay 4 in results) was performed. Last attempted SDS-PAGE by Drew, resulting in another failure. The ligation reaction was corrected; transformation was a success. Restriction digest was positive.

### **Week 18 – October 1st-5th**

Initial sequencing was promising, but didn’t run the full length of the gene. Follow-up sequencing was performed and confirmed a positive cloning.

### **Week 19 – October 8th-12th**

Nic ran miniprep for the new pSB1C3\_TPS4-CO. Plenty of DNA was pipetted into the 96 well submission plate and dried overnight. Our part was submitted by Nic on the 9th and the package was successfully delivered to iGEM HQ on the 15th.

### **Week 20 – October 15th-19th**

All wet lab work is completed by this point. The team is frantically trying to finish the wiki.

# Thanks to Our Sponsors

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# Team:UNebraska-Lincoln/Experiments

# UNL 2018 Improving Early Detection of The Emerald Ash Borer

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[Team](https://2018.igem.org/Team%3AUNebraska-Lincoln/TeamLanding)

[Team](https://2018.igem.org/Team%3AUNebraska-Lincoln/Team)
[Collaborations](https://2018.igem.org/Team%3AUNebraska-Lincoln/Collaborations)

[Project](https://2018.igem.org/Team%3AUNebraska-Lincoln/ProjectLanding)

[Description](https://2018.igem.org/Team%3AUNebraska-Lincoln/Description)
[Design](https://2018.igem.org/Team%3AUNebraska-Lincoln/Design)
[Experiments](https://2018.igem.org/Team%3AUNebraska-Lincoln/Experiments)
[Notebook](https://2018.igem.org/Team%3AUNebraska-Lincoln/Notebook)
[InterLab](https://2018.igem.org/Team%3AUNebraska-Lincoln/InterLab)
[Contribution](https://2018.igem.org/Team%3AUNebraska-Lincoln/Contribution)
[Model](https://2018.igem.org/Team%3AUNebraska-Lincoln/Model)
[Results](https://2018.igem.org/Team%3AUNebraska-Lincoln/Results)
[Attributions](https://2018.igem.org/Team%3AUNebraska-Lincoln/Attributions)

[Parts](https://2018.igem.org/Team%3AUNebraska-Lincoln/PartsLanding)

[Basic Parts](https://2018.igem.org/Team%3AUNebraska-Lincoln/Basic_Part)

[Safety](https://2018.igem.org/Team%3AUNebraska-Lincoln/Safety)

[Human Practices](https://2018.igem.org/Team%3AUNebraska-Lincoln/HumanLanding)

[Silver HP](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Silver)
[Integrated and Gold](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Gold_Integrated)

# EXPERIMENTS

---

---

### ›LB Broth Preparation

#### Materials

* LB Broth powder (Lennox)
* 250 mL media bottle
* 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 (Liquids cycle).
6. Retrieve and let cool to room temperature before use.

### ›Terrific Broth Preparation

#### Materials

* 500 mL media bottle
* Tryptone powder
* Yeast extract
* Autoclaved glycerol
* Nanopure water
* Phosphate buffer

#### Procedure

1. Weigh 10 grams of tryptone powder.
2. Weigh 12 grams of yeast extract.
3. Mix and add 450 mL of nanopure water.
4. Add ~5 mL of autoclaved glycerol if desired.
5. Autoclave, retrieve, and let solution completely cool.
6. Add 50 mL of phosphate buffer. **NOTE: Phosphate buffer is prepared by adding 4.65g monopotassium phosphate and 25g dipotassium phosphate into 200 mL PCR-grade water.**

### ›Fresh Agar Plate Preparation

#### Materials

* ~ 24 petri dishes
* 500 mL media bottle
* Corresponding amount of 100x antibiotic stock solutions (500 µL)

#### Procedure

1. Obtain and weigh 25g LB Broth powder/L of nanopure water.
2. Obtain and weigh 15g agar powder/L of nanopure water.
3. Mix into respective volume of nanopure water.
4. Autoclave solution for 1 hour (the first setting).
5. Retrieve solution and wait to cool.
6. Add any necessary antibiotics, typically chloramphenicol (Cm) or ampicillin (Amp) stock solution (1000x) with 1-to-1000 dilution.
7. Evenly pour solution into properly labeled petri dishes.
8. Allow solution to then solidify.
9. Keep agar plates in 4 °C refrigerator.

### ›Plasmid Mini-Prep

#### Materials

* 1.5 mL eppendorf tubes
* Thermo Scientific Plasmid Miniprep Kit
* P1, P2, and P3 buffers
* Endo-wash buffer
* Zyppy-wash buffer
* PCR-grade water

#### Procedure

1. Centrifuge cell culture for 10 min at 4°C at 5,000rpm.
2. Pour out the LB Broth Supernatant into bleach solution.
3. Add 200 μL P1 buffer and resuspend pellet.
4. Transfer into a fresh 1.5 mL eppendorf tube.
5. Add 200 μL P2 buffer, mix by slowly inverting until transparent
6. Get P3 buffer from fridge and add 400 μL. Invert tube 2-4 times and incubate for 1 minute.
7. Centrifuge tube at max speed for 5 minutes.
8. Transfer all the supernatant into the zymo-spin column immediately after.
9. Place the column into a collection tube and centrifuge at 16,000 g for 30 seconds. Discard the flow-through and place the column back into the same collection tube.
10. Add 200 µL of Endo-wash buffer to the column. Centrifuge at 16,000 g for 30 seconds.
11. Add 400 µL of Zyppy-wash buffer to the column. Centrifuge at 16,000 g for 30 seconds.
12. Centrifuge for two minutes at 21,000 g.
13. Transfer the column into a clean 1.5 mL eppendorf tube, then add 30 µL of PCR-grade water directly to the column matrix.
14. Let it stand for one minute at room temperature.
15. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.
16. Typically take Nanodrop measurement right after.

### ›Polymerase Chain Reaction

#### Materials

* PCR tubes
* dNTPs
* 10x PCR buffer
* MgSO4
* Template DNA
* Primers
* KOD Polymerase
* PCR-grade water
* Thermocycler

#### Procedure

1. For just one 25 µL PCR mix, the following table was applied. The reaction size was typically scaled up to meet specific experiment demand.

   | **Materials** | **Volume (µL)** |
   | --- | --- |
   | dNTPs (10mM) | 2.5 |
   | 10x PCR buffer | 2.5 |
   | MgSO4 | 1.5 |
   | Template (~10ng/µL) | 1 |
   | Primers (10µM) | 0.75 each |
   | KOD Polymerase | 0.5 |
   | PCR-grade water | 16.25 |
2. Place the prepared reaction tubes into the thermocycler and run program tailored to the target product size. Our target product size was always between 1000-3000 bp.

   | **Step** | **Target Size** | **Target Size** | **Target Size** | **Target Size** |
   | --- | --- | --- | --- | --- |
   | **-** | < 500 bp | 500-1000 bp | 1000-3000 bp | > 3000 bp |
   | **Polymerase Activation** | 95°C for 2 min | 95°C for 2 min | 95°C for 2 min | 95°C for 2 min |
   | **Denaturing** | 95°C for 20s | 95°C for 20s | 95°C for 20s | 95°C for 20s |
   | **Annealing** | Lowest Primer Tm for 10s (~65°C) | Lowest Primer Tm for 10s (~65°C) | Lowest Primer Tm for 10s (~65°C) | Lowest Primer Tm for 10s (~65°C) |
   || **Extension** | 70°C for 10s/kb | 70°C for 15s/kb | 70°C for 20s/kb | 70°C for 25s/kb |
   | --- | --- | --- | --- | --- |
   | **Halt** | 4°C for infinity | 4°C for infinity | 4°C for infinity | 4°C for infinity |

### ›Standard 1% Gel Electrophoresis

#### Materials

* Agarose powder
* TAE buffer
* Erlenmeyer flask
* Ethidium Bromide
* Casting tray
* Sub-Cell apparatus
* 2-log 10kb ladder
* 6x Loading Dye
* DNA Product

#### 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. Carefully remove the comb.
9. Load ~5 µL of the ladder into the leftmost well.
10. Mix the loading dye with the DNA sample to the proper concentration and then load into well(s).
11. Once your samples are loaded place the lid on the apparatus and run the gel at 80 volts for 1 hour.
12. Carefully remove gel to image.

### ›Gel DNA Recovery

#### Materials

* Zymoclean™ Gel DNA Recovery Kit
* ADB Buffer
* Wash Buffer
* Hot water bath
* Eppendorf tubes
* PCR-grade water

#### Procedure

1. Weigh the piece of cut gel, then add 3 volumes of ADB buffer to each volume of gel. (max volume in tube = 750 µL)
2. Perform short centrifuge
3. Incubate the 1.5 mL eppendorf tube in a 55°C water bath for 5-10 minutes.
4. Use vortex to mix well.
5. Add the melted agarose solution into a Zymo-spin column and place into a collection tube.
6. Centrifuge for 30 seconds at 16,000 g. Empty the collection tube whenever necessary.
7. Add 200 µL of Wash buffer to the column and centrifuge for 30 seconds.
8. Repeat step 7.
9. Centrifuge at max speed for 2 minutes.
10. Place Zymo-Spin column into a new 1.5 mL tube.
11. Add 8-12 µL of PCR-grade water directly to the column matrix.
12. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.
13. Typically take Nanodrop measurement right after.

### ›Sequence and Ligation Independent (SLIC) Cloning

#### Materials

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

#### Procedure

1. A table is offered below for guidance. 150ng 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 (1:2 molar ratio).
3. Calculate the amount of PCR-grade 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 (23 °C for 30 min; 75 °C for 15 min; 37 °C for 30 min; hold at 4 °C)
9. Transform product.

- For just one 25 µL PCR mix, the following table was applied. The reaction size was typically scaled up to meet specific experiment demand.

  | **Materials** | **Volume** |
  | --- | --- |
  | Vector | ~150ng |
  | Insert | 1:2 vector to insert molar ratio |
  | 10x Buffer 2.1 (NEB) | 2 µL |
  | T4 DNA Polymerase (1:6 dilution in 2.1 buffer) | 1 µL |
  | PCR-grade water | Amount necessary for 20 µL reaction |

### ›Transformation

#### Materials

* Genehog Chemically Competent Cells
* Hot water bath
* Eppendorf tubes
* Agar plates with antibiotic
* 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 30-60 seconds.
4. Put cells back on ice for 2 minutes. Sterilize work area and pipet in this time.
5. Add 1 mL of LB broth to the mixture.
6. Leave the cells at 37°C for one hour in shaker. Meantime, prewarm agar plates.
7. Plate cells using beads (volume varies slightly).
8. Incubate the plate overnight at 37°C.
9. Look for colonies.

### ›Restriction Enzyme Digest

#### Materials

* Restriction enzymes (variable)
* 10x 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. *Xba*I and *Spe*I).
4. Gently mix together. Total reaction volume is 20 µL.
5. Place in incubator for 1½ hours at 37°C.
6. Run gel electrophoresis and perform gel DNA recovery on product.
7. Store in 4°C chamber.

### ›Subcloning (pSB1C3 vector backbone)

#### Materials

* T4 Ligase
* 10x Ligase buffer
* HF restriction enzymes
* PCR tubes
* PCR-grade water

#### Procedure

1. Perform restriction enzyme digest on vector using *EcoR*I and *Pst*I.
2. Perform restriction enzyme digest on DNA insert using *EcoR*I and *Pst*I.
3. Recover DNA after gel electrophoresis.
4. 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.
5. Load the cut vector and insert into PCR tube.
6. Add enough 10x Ligase Buffer so that the buffer is one-tenth the total reaction volume.
7. Add 0.5 µL of T4 DNA Ligase into the PCR tube.
8. Prepare a negative control by replacing the cut DNA insert portion with water.
9. Place PCR tubes into thermocycler and run the ligation protocol.
10. Transform product.

# Thanks to Our Sponsors

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# Team:UNebraska-Lincoln/Results

# UNL 2018 Improving Early Detection of The Emerald Ash Borer

[![](https://static.igem.org/mediawiki/2018/thumb/9/94/T--UNebraska-Lincoln--fullLogo_2018.png/633px-T--UNebraska-Lincoln--fullLogo_2018.png)](https://2018.igem.org/Team%3AUNebraska-Lincoln)

[Team](https://2018.igem.org/Team%3AUNebraska-Lincoln/TeamLanding)

[Team](https://2018.igem.org/Team%3AUNebraska-Lincoln/Team)
[Collaborations](https://2018.igem.org/Team%3AUNebraska-Lincoln/Collaborations)

[Project](https://2018.igem.org/Team%3AUNebraska-Lincoln/ProjectLanding)

[Description](https://2018.igem.org/Team%3AUNebraska-Lincoln/Description)
[Design](https://2018.igem.org/Team%3AUNebraska-Lincoln/Design)
[Experiments](https://2018.igem.org/Team%3AUNebraska-Lincoln/Experiments)
[Notebook](https://2018.igem.org/Team%3AUNebraska-Lincoln/Notebook)
[InterLab](https://2018.igem.org/Team%3AUNebraska-Lincoln/InterLab)
[Contribution](https://2018.igem.org/Team%3AUNebraska-Lincoln/Contribution)
[Model](https://2018.igem.org/Team%3AUNebraska-Lincoln/Model)
[Results](https://2018.igem.org/Team%3AUNebraska-Lincoln/Results)
[Attributions](https://2018.igem.org/Team%3AUNebraska-Lincoln/Attributions)

[Parts](https://2018.igem.org/Team%3AUNebraska-Lincoln/PartsLanding)

[Basic Parts](https://2018.igem.org/Team%3AUNebraska-Lincoln/Basic_Part)

[Safety](https://2018.igem.org/Team%3AUNebraska-Lincoln/Safety)

[Human Practices](https://2018.igem.org/Team%3AUNebraska-Lincoln/HumanLanding)

[Silver HP](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Silver)
[Integrated and Gold](https://2018.igem.org/Team%3AUNebraska-Lincoln/HP/Gold_Integrated)

---

# Results

---

---

### **Overview:**

We are excited to present the following results:

* Successfully cloned and fully sequenced codon-optimized TPS4-B73 gene (7-epi-sesquithujene synthase gene) into the vector p100169.
  Also successfully cloned and fully sequenced non-codon optimized TPS4-B73 gene into the same vector.
* Confirmed FPP production and tested our sesquiterpene production assay using ADS (amorpha-4,11-diene synthase).
* Observed in vivo production of sesquiterpenoid, including target molecule 7-epi-sesquithujene, by codon-optimized TPS4-B73 and identified major products.
* Quantified sesquiterpene production in E. coli culture expressing the codon-optimized TPS4-B73.

### Cloning:

We first cut out the ADS gene from p100169. To clone TPS4 into the vector, we used Sequence and Ligation Independent Cloning (SLIC).
On our third SLIC attempt, we isolated the plasmid from six colonies and performed a restriction digest to identify if any of the constructs were correct.

### Fig 0.1: Gel electrophoresis of digested SLIC products

![](https://static.igem.org/mediawiki/2018/4/49/T--UNebraska-Lincoln--Results_fig01_2018.png)

The fourth clone had the expected digest pattern of two bands, so we proceeded to fully sequence the plasmid around our part to confirm our construct pTPS4.

We used restriction cloning to clone the codon-optimized TPS4. There were many colonies on the control empty vector plate, so we chose six colonies to test the ligation product with a restrictive digest.

### Fig 0.2: Gel electrophoresis of digested ligation products

![](https://static.igem.org/mediawiki/2018/7/7e/T--UNebraska-Lincoln--Results_fig02_2018.png)

Five of the colonies showed the correct digestive pattern. We fully sequenced the plasmid of the first colony around our part to confirm our construct pTPS4CO.

### Confirming FPP production:

We ran our sesquiterpene production assay 4 times, each time with different growth variables.
The strains tested each had the FPP producing p100167 and a sesquiterpene synthase plasmid.
For the first 3 assays, we tested p100169 (pADS), pTPS4, and pTPS4CO.
ADS is the well-characterized amorpha-4,11-diene synthase, so we knew that if the pADS cultures produced amorpha-4,11-diene,
then the mevalonate-dependent pathway was working and FPP was successfully being produced. This would show that our sesquiterpene production was not being bottlenecked by FPP production.
We took OD600 measurements and took octane samples to measure the culture growth and sesquiterpene production. The octane samples were analyzed through gas chromatography-mass spectrometry.

In each assay, we observed the presence of amorpha-4,11-diene in our pADS cultures.

### Fig 1: Gas chromatogram of the first assay 24 hour sample showing the presence of the amorpha-4,11-diene peak with a retention time of 7.684 minutes

![](https://static.igem.org/mediawiki/2018/b/b4/T--UNebraska-Lincoln--resultsfig_1_ads_1-24_2018.png)

### Fig 2: Standard mass spectrum of amorpha-4,11-diene from the NIST library

![](https://static.igem.org/mediawiki/2018/e/e7/T--UNebraska-Lincoln--resultsfig_2_ads_standard_2018.png)

### Fig 3: Mass spectrum of produced amorpha-4,11-diene

![](https://static.igem.org/mediawiki/2018/e/e9/T--UNebraska-Lincoln--resultsfig_3_ads_mass_spec_2018.png)

The mass fragmentation pattern is unique to each molecule, like a molecular fingerprint.
The mass detection range on the instrument we used had a lower limit of 50 m/z, so any characteristic peaks below 50 m/z will not appear on our samples.
Comparison of the mass spectrum of our product to the standard identifies the product as the expected amorpha-4,11-diene.
The presence of amorphadiene showed that the mevalonate-dependent pathway was functional and was producing sufficient amounts of FPP for sesquiterpene production.

### pTPS4CO product identification:

In the first assay, our pTPS4 and pTPS4CO cultures were tested, but only pTPS4CO produced sesquiterpenoid products.
Since FPP was being produced under these conditions as shown in the pADS culture, the failure of pTPS4 was not a problem of FPP production.
We were able to identify the major products of pTPS4CO to determine functionality.

### Fig. 4: Gas chromatogram of the 120 hour sample of pTPS4CO sesquiterpene products. 7-epi-sesquithujene had a retention time of 6.689 minutes, and beta-bisabolene had a retention time of 7.989 minutes.

![](https://static.igem.org/mediawiki/2018/e/ed/T--UNebraska-Lincoln--resultsfig_4_tps4co_gc_2018.png)

### Fig. 5: Standard mass spectrum of 7-epi-sesquithujene from the NIST library

![](https://static.igem.org/mediawiki/2018/2/28/T--UNebraska-Lincoln--resultsfig_5_7-epi_std_2018.png)

### Fig. 6: Mass Spectrum of produced 7-epi-sesquithujene

![](https://static.igem.org/mediawiki/2018/7/73/T--UNebraska-Lincoln--resultsfig_6_7-epi_2018.png)

The mass detection range on the instrument we used had a lower limit of 50 m/z, so any characteristic peaks below 50 m/z will not appear on our samples. Comparison of the mass spectrum with the standard identifies the product with retention time 6.689 minutes as 7-epi-sesquithujene, which means that our pathway and part were functional.
We were also able to identify the other major product, with retention time 7.989 minutes, as beta-bisabolene.

### Fig. 7: Standard mass spectrum of beta-bisabolene

![](https://static.igem.org/mediawiki/2018/2/2a/T--UNebraska-Lincoln--resultsfig_7_std_beta_bisabolene_2018.png)

### Fig. 8: Mass spectrum of produced beta-bisabolene

![](https://static.igem.org/mediawiki/2018/3/35/T--UNebraska-Lincoln--resultsfig_8_beta_bisabolene_2018.png)

The mass detection range on the instrument we used had a lower limit of 50 m/z, so any characteristic peaks below 50 m/z will not appear on our samples. Comparison of the mass spectrum to the standard identifies the product as beta-bisabolene.
This was further evidence that our part was functioning correctly in vivo, since both 7-epi-sesquithujene and beta-bisabolene had been predicted as the major products in in vitro experiments (Kollner, 2004).

### Product Quantification:

After we had identified that our pathway was working and our part was producing the desired product, we developed a product quantification method.
7-epi-sesquithujene is commercially unavailable, so we could not use it as an analytical quantification standard; instead, we used the sesquiterpene (-)-trans-caryophellene as an internal standard.
It is not one of the potential products of 7-epi-sesquithujene synthase (Kollner et al, 2004), and it has a retention time of 7.210, which is separated from the products we wished to quantify.
To calculate the concentrations of our products, we used the internal standard method developed by Martin et al in 2001. The concentrations of 7-epi-sesquithujene and beta-bisabolene
are reported as (-)-trans-caryophellene equivalents based on the relative abundance of the 204, 161, 119, and 105 ions to all other ions for each product.
These ions are the molecular ion and other distinctive ions to sesquiterpenes. The ratios were 10.9%, 20.1%, and 9.05% for (-)-trans-caryophellene, 7-epi-sesquithujene, and beta-bisabolene respectively.

### Fig. 9: Selective ion search for the 204, 161, 119, and 105 ions

![](https://static.igem.org/mediawiki/2018/6/6f/T--UNebraska-Lincoln--resultsfig_9_selective_ion_2018.png)

Using this method, we were able to quantify the amounts of products in each of our 4 assays and compare growing conditions to production.

### Assay Growth Conditions:

Our first assay had an initial OD600 of 0.02, and the cultures were induced at an OD600 ranging from 0.1-0.4 for the three cultures.
Unfortunately, due to other students in the lab, the shaker speed was not kept constant at 150 rpm, but for hours 48-120 was set at 250 rpm.
The assay went for 120 hours instead of 72 hours due to lab accessibility issues. For this and following assays, the cultures were induced and octane was added at the zero hour.

Our second assay had a higher initial OD600 of 0.07, and the cultures were induced at a much higher OD600 ranging from 1-4 for the three cultures.
Again, due to other students in the lab, the shaker speed was not kept constant at 150 rpm and was instead set at 250 rpm for the first 24 hours, which caused culture growth to differ from how we had expected.
They were grown for 48 hours.

Our third assay had an initial OD600 of 0.05 and an induction OD600 of 0.15. The shaker was set at 150 rpm throughout the experiment, and the cultures were grown for 72 hours.

Each of these three assays tested pADS, pTPS4, and pTPS4CO. Our fourth assay tested six biological replicates of pTPS4CO.
They had an initial OD600 of 0.05 and an induction OD600 ranging from 0.15-0.53. They were grown for 49 hours.

### Assay Production Results:

### Fig. 10: pADS growth and amorpha-4,11-diene production

![](https://static.igem.org/mediawiki/2018/f/f7/T--UNebraska-Lincoln--results_fig_10_2018.png)

Amorphadiene was produced under all three growing conditions, so FPP was being produced in all 3 experiment conditions.
The results show that pADS favored the growing conditions with induction at the higher optical density.

### Fig 11: pTPS4CO growth and production

![](https://static.igem.org/mediawiki/2018/9/95/T--UNebraska-Lincoln--figure11.png)

The results show that 7-epi-sesquithujene was produced under all growing conditions.
Unlike pADS, pTPS4CO but was not optimal when induced at a higher optical density.
The results show that the production of 7-epi-sesquithujene can vary widely with different growing conditions.

Non-codon optimized pTPS4 was also tested in assays 1-3. It did not produce any sesquiterpenes in assay 1, but in assays 2 and 3 it did produce sesquiterpenes;
however, we did not move forward in further testing due to time constraints.
We chose to do further testing on the codon-optimized variant because it had consistently produced sesquiterpenes in each assay, albeit of varying amounts.
We wished to test whether pTPS4CO production was consistent under identical growing conditions.

In our fourth assay, we tested six biological replicates of pTPS4CO. Unfortunately, an error was made in measuring the OD600 at the 29 hour time point for replicates 2,3, and 4.

### Fig. 12: Growth and production of correctly measured TPS4CO replicates 1, 5, and 6

![](https://static.igem.org/mediawiki/2018/c/ce/T--UNebraska-Lincoln--newResultsfigure12_2018.png)

### Fig. 13: Growth and production of biological replicates 2, 3, and 4 with missing OD600 data point at 29 hours

![](https://static.igem.org/mediawiki/2018/d/dc/T--UNebraska-Lincoln--newResultsfigure13_2018.png)

The biological replicates were grown under the same conditions; however, the resulting concentrations of 7-epi-sesquithujene varied more widely than our previous results testing growing conditions.
Under these identical conditions, 7-epi-sesquithujene production ranged from 0.52 to 92.78 mg/L.
We concluded that there must be a production variable that we have not discovered yet that is causing the variance.

We also quantified beta-bisabolene production in our biological replicates of pTPS4CO. Literature of in vivo experiments comparing the relative abundance of 7-epi-sesquithujene
synthase reported that beta-bisabolene should be the major product, constituting 29.1% of total products, with 7-epi-sesquithujene as the second major product, constituting 24.4% of total products (Kollner, 2004). This produced a 7-epi-sesquithujene to beta-bisabolene ratio of 0.83.

### Fig. 14: Comparison of 7-epi-sesquithujene production to beta-bisabolene production at the 49 hour time point of assay 4

![](https://static.igem.org/mediawiki/2018/0/05/T--UNebraska-Lincoln--results_fig_14_2018.png)

These results show that 7-epi-sesquithujene was our major product instead of beta-bisabolene. Although the final concentrations varied in magnitude,
each replicate produced slightly more 7-epi-sesquithujene than beta-bisabolene. The observed ratio of 7-epi-sesquithujene to beta-bisabolene was 1.05 with a standard deviation of 0.03. This was different than the literature had reported,
where the ratio of 7-epi-sesquithujene to beta-bisabolene was 0.83 and beta-bisabolene was clearly the major product. This showed that either codon optimization, performing the experiment in vivo,
or different growth conditions could change the product profile of 7-epi-sesquithujene synthase.

### Other experiments:

We attempted to do an SDS-PAGE several times to confirm the presence of 7-epi-sesquithujene synthase, but we had trouble with the procedure.
New technical difficulties occurred each time we ran the procedure, so unfortunately we ran out of time to get conclusive results.

A preliminary purification experiment was done to attempt to purify amorpha-4,11-diene, but we ran out of time to get conclusive results and perform the procedure to purify 7-epi-sesquithujene.

### Other experiments:

* Purify 7-epi-sesquithujene to perform nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR) analysis to further confirm product identity
* Repeat sesquiterpene production assays with a higher sample collection rate to identify production variables
* Optimize the assay procedure to increase 7-epi-sesquithujene yields

### Considerations for replicating experiments:

* In our experiments, pTPS4CO always produced some amount of sesquiterpene products. Replicate experiments should always produce products, but the yields may vary until further production variables are identified.

---

Works Cited

* Martin, V. J.; Yoshikuni, Y.; Keasling, J. D. The in vivo synthesis of plant sesquiterpenes by *Escherichia coli. Biotechnology and Bioengineering* **2001**, 75(5), 497–503.
* Köllner, T. G.; Schnee, C.; Gershenzon, J.; Degenhardt, J. The variability of sesquiterpenes emitted from two *Zea mays cultivars is controlled by allelic variation of two terpene synthase
  genes encoding stereoselective multiple product enzymes. *The Plant Cell* **2004**, 16(5), 1115–1131.*

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