iGEM 2021 — Team WrightState

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# Team:WrightState/Notebook

Team

![](https://static.igem.org/mediawiki/2021/1/1b/T--WrightState--logo.png)

# WrightState

Building a better organic machine

## Notebook

* July 6 and 8 – Team introduction to Synthetic Biology
* July 13 and 15 – Team reviews Synthetic Biology information and begins brainstorming.
* July 20 and 22 – Team research on a-terpineol
* July 27 and 29 – Team begins in wet lab and other members continue research
* August 4 – Team meets with mentors to discuss current progress
* August 3 and 5 – Team works on Outreach activity for Chabad of Greater Dayton
* August 9 – Team does Strawberry DNA extraction with kids at Chabad of Greater Dayton
* August 11 – Team meets with mentors to discuss progress
* August 17 and 19 – Team begins posting more to social media (Meet the Team and Synbio Saturdays)
* August 18 – Team meets with mentors to discuss science (issue with digesting JBEI)
* August 24 and 26 – Team prepares to host an Ohio meetup with other iGEM teams
* August 25 – Team meets with mentors to discuss their experiences and what to expect at an iGEM meetup
* August 31 and September 3 – Science slows for a short time as the team begins school but work on wiki drafts and content begins
* September 1 – Team meets with mentors to discuss the promotional video and what should look like.
* September 7 and 9 – Team Discusses the next presentation and what order things should be presented in
* September 8 – Team discusses presentation outline with mentors
* September 14 and 16 – Team prepares presentation and practices
* September 15 – Team practices presentation with mentors to get feedback
* September 21 and 23 – Team works on wiki content and outreach
* September 22 – Team meets with Dr. Michael Miller to discuss his expertise in the field of monoterpene production
* September 28 and 30 – Team works on wiki content and EPA discussions
* October 5 and 7 – Team works on presentation for Dr. Gallivan
* October 6 – Team discusses presentation with mentors along with wiki content
* October 12 and 14 – Team finalizes presentation for Dr. Gallivan and makes outline for final iGEM presentation
* October 13 – Team gives presentation

### Our address

**Wright State University**
**3640 Colonel Glenn Hwy.,
Dayton, OH 45435**
937-775-1000
Email Us

### Social media

@igemwrightstate

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# Team:WrightState/Engineering

Engineering Success

![](https://static.igem.org/mediawiki/2021/1/1b/T--WrightState--logo.png)

# WrightState

Building a better organic machine

## Engineering

Our approach in biomanufacturing α-Terpineol was a two plasmid system that would be able to utilize a native yeast pathway to convert Acetyl-CoA all the way to α-Terpineol . This native yeast pathway is called the Mevalonate pathway and is normally used for momoterpene production. However, we decided to utilize this system in E. coli rather than yeast. This decision was made due to natural inhibitors that exist in yeast that may cause a low yield. The thought was to remove the inhibitors and get a much higher production of our monoterpene.

![Project](https://static.igem.org/mediawiki/2021/0/01/T--WrightState--ES1.png)

![Project](https://static.igem.org/mediawiki/2021/0/0f/T--WrightState--ES2.png)

Design of Dual Plasmid System

The first plasmid (JBEI-3085) contains the MEV pathway and produces IPP and DMAPP

![Project](https://static.igem.org/mediawiki/2021/b/b2/T--WrightState--ES3.png)

The second plasmid (pTrc\_GppsGb\_VvTs) was purchased from Addgene (#50603) and modified to produce α -Terpineol

![Project](https://static.igem.org/mediawiki/2021/4/4b/T--WrightState--ES4.png)

### \*Addgene plasmid (#50603) prior to being digested and recloned with newly designed part (BBa\_K4078003)

![Project](https://static.igem.org/mediawiki/2021/c/ca/T--WrightState--ES5.png)

### \*Cloned to include GPPS/αTS geneblock from IDT

\*Our GPPS (BBa\_K2753002) was used from a past iGEM team (Great Bay). This newly constructed plasmid contains a LacI repressor gene and is inducible by IPTG\*

**Build**

To build our plasmids we used transformation methods with MG1655 competent cells and sequenced the resulting mini-preps to confirm prior to moving forward. The building process took the most time as we were having issues with digests and verifying what we were sent is correct. Numerous gels were run, and many digests were performed but after that was figured out we were ready for production assays.

**Test**

In order to test production, we ran production assays where 5mL cultures were grown to an OD600 of 0.8. 1mL of dodecane was added along with the given concentration of IPTG and allowed to grow for 72 hours. To extract, 4mL of ethyl acetate was added and the organic layer was harvested, the samples were then quantified with GCMS.

![Project](https://static.igem.org/mediawiki/2021/8/8a/T--WrightState--ES6.png)

Quantification by GC/MS

![Project](https://static.igem.org/mediawiki/2021/6/6a/T--WrightState--ES7.png)

We believe that the production levels that we obtained may be too low to be detected by GC/MS. Though as it is being shown, there is no production of α -Terpineol at the moment and something that can be optimized and worked on in the years to come.

**Learn**

The experiments that followed the discovery of zero production included a few different steps. The first was varying the concentrations in which we induced production with IPTG to see if the problem was the use of note enough or far too much. We also spoke to an expert in the field, Dr. Michael Miller, who worked with terpene production for several years, and he suggested that switching the order of the GPPS and the αTS may affect production.

In the future, we plan to use different enzymes, try out different RBS as well as Bacterial strains that may be more effective. Though it is still a work in progress, we are continually learning from our results and reevaluating.

**Sources**

Sarria, S., Wong, B., Martín, H. G., Keasling, J. D., & Peralta-Yahya, P. (2014). Microbial synthesis of pinene. ACS Synthetic Biology, 3(7), 466–475. https://doi.org/10.1021/sb4001382

Alonso-Gutierrez, J., Chan, R., Batth, T. S., Adams, P. D., Keasling, J. D., Petzold, C. J., & Lee, T. S. (2013). Metabolic engineering of escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, 19, 33–41. https://doi.org/10.1016/j.ymben.2013.05.004

### Our address

**Wright State University**
**3640 Colonel Glenn Hwy.,
Dayton, OH 45435**
937-775-1000
Email Us

### Social media

@igemwrightstate

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

# Team:WrightState/Experiments

Team

![](https://static.igem.org/mediawiki/2021/1/1b/T--WrightState--logo.png)

# WrightState

Building a better organic machine

## Experiments

### Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010)

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve. Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as it contains RNase A. Note: unlike other commercial kits, all wash steps are required.

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through.
10. Add ≥ 30 μl nuclease free water to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

### Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020)

All centrifugation steps should be carried out at 16,000 x g (around 13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.

1. Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 ml microcentrifuge tube and weigh the gel slice.
2. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 μl buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present in the workflow.
3. Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes).
4. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
6. Repeat wash (Step 5).
7. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
8. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

### Sample Restriction Enzyme Digestion Protocol (NEB)

Steps:

1. Set up reaction as follows:

![Project](https://static.igem.org/mediawiki/2021/7/7a/T--WrightState--DigFigure.png)

2. Incubate at 37°C for 5–15 minutes as NcoI-HF is Time-Saver qualified.

### NEBuilder HiFi DNA Assembly Chemical Transformation Protocol

1.) Thaw competent cells (50 uL/tube)
2.) Add 2 uL of chilled assembly product to the competent cells. Mix gently by pipetting up and dow or by flicking the tube 4-5 times. Do not vortex.
3.) Place mixture on ice for 30 min. Do not mix.
4.) Heat shock at 42oC for 30 seconds. Do not mix.
5.) Transfer tubes to ice for 2 min.
6.) Add 950 uL of room temp SOC stable outgrowth medium to tube.
7.) Incubate the tube at 37C for 60 min. Shake at 250 rpm.
8.) Warm selection plates to 37C
9.) Spread 100 uL of cells onto the selection plate
10.) Incubate ON at 37C

### Transformation Protocol for MG1655 cells

1. Incubate DNA with comp cells for 30 - 45 min on ice. (With the low competency, I would not go shorter than 30 min).
2. Heat shock at 42 C for 30 sec.
3. Put transformation mix back on ice for 1-2 min.
4. Add 300 uL SOC (or LB) into transformation mix.
5. Recover transformation cells at 37 C, shaking, for 1-2 hrs.
6. Plate transformation on appropriate selection plates. Plate out transformation on 3 plates - 50 uL, 100 uL, and the remaining transformation.

### EZ rich media Protocol

![Project](https://static.igem.org/mediawiki/2021/7/7e/T--WrightState--EZMedia.png)

### HiFi DNA Assembly

1. Set up the following reaction on ice:

![Project](https://static.igem.org/mediawiki/2021/2/29/T--WrightState--HifiDNAAssembly.png)

2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.

3. Transform NEB 5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μl of the chilled assembled product, following the transformation protocol.

### α-Terpineol Production Assay Protocol

1. Inoculate 5 mL EZ-rich media with transformed MG1655 cells and add given amount of antibiotics
2. Incubate culture in shaker at 37oC and 250 rpm until the culture reaches an OD600 of 0.8
3. Induce culture with 1 mM IPTG and overlay with 20% dodecane
4. Incubate culture for 72 hours at 30oC and 250 rpm
5. Add 4 mL ethyl acetate and briefly vortex
6. Extract organic layer and send off for GC/MS analysis and quantification

### Our address

**Wright State University**
**3640 Colonel Glenn Hwy.,
Dayton, OH 45435**
937-775-1000
Email Us

### Social media

@igemwrightstate

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

# Team:WrightState/Results

Team

![](https://static.igem.org/mediawiki/2021/1/1b/T--WrightState--logo.png)

# WrightState

Building a better organic machine

## Results

### Goal:

* Construct dual plasmid system in bacteria capable of producing α-terpineol

### Approach:

After doing literature research, we discovered a series of pathways that if connected through enzymatic activity had the capability of producing α-terpineol. The first pathway is known as the yeast Mevalonate (MEV) pathway. This pathway starts with acetyl-CoA as its substrate and though a series of enzymatic conversions produces IPP & DMAPP. The second pathway is the basic monoterpene production pathway. This pathway is able to take IPP & DMAPP and convert these into α-terpineol via a two-step conversion utilizing geranyl pyrophosphate synthase (GPPS) & α-terpineol synthase (αTS) as shown in the figure below. We used the αTS from the organism Vitis vinifera in the following experiments, hereafter referred to as VvTs.

\*The αTS used in the following experiments is referenced as VvTs as this αTS came from the organism Vitis vinifera\*

![Project](https://static.igem.org/mediawiki/2021/b/b4/T--WrightState--Results1.png)

In our dual plasmid model, the first plasmid was readily available via Addgene known as JBEI-3085 (#87950). This plasmid contains the enzymes found in the yeast MEV pathway. The second plasmid also known as pTrc\_GppsGB\_VvTs had to be constructed to contain a GPPS & the Vitis vinifera αTS (VvTs). In order to accomplish this, we were able to digest and isolate a backbone from our source plasmid (pTrc\_GPPS\_LS) via gel purification that has been previously used for other monoterpene production as shown in the figure below. The 4.1kb backbone (indicated in the red box) was successfully gel-purified from the excised 2.6kb insert (bottom band).

![Project](https://static.igem.org/mediawiki/2021/f/f5/T--WrightState--Results2.png)

### Gel purification of backbone

After successful gel purification of our desired backbone, we performed NEBuilder HiFi DNA assembly with our designed insert and sequentially transformed this plasmid (pTrc\_GppsGb\_VvTs) into competent NEB 5-α cells.

![Project](https://static.igem.org/mediawiki/2021/3/3b/T--WrightState--Results3.png)

### Transformation of pTrc\_GppsGb\_VvTs into NEB 5- α cells

A miniprep kit was used to extract and purify our constructed plasmid for transformation into MG1655 E. coli cells. To confirm cloning of our plasmid we conducted a restriction enzyme digest and sent off our plasmid for sequencing. Once confirmed, we double transformed pTrc\_GppsGb\_VvTs along with JBEI-3085. A restriction enzyme digest was also used to confirm these transformations as shown in the figure below.

![Project](https://static.igem.org/mediawiki/2021/a/a1/T--WrightState--Results4.png)

### Verification of double transformation of pTrc\_GppsGb\_VvTs & JBEI-3085 into MG1655 cells

With the confirmation of these transformations, we were able to proceed with production trials of α-terpineol. The production assays were ran by inducing our bacterial cultures with IPTG in glucose rich media known as EZ-rich media with a dodecane overlay followed by an incubation period of 72 hours while shaking at 250rpm. After induction, any α-terpineol produced should be in the organic dodecane layer. The samples were extracted by adding ethyl acetate and removing only the top organic layer for GC/MS quantification.

Before production trials were run, the extraction method and limit of detection for our GC/MS approach were evaluated. The extraction method was verified by spiking in a known mount of α-terpineol to blank samples. The samples were then extracted and sent for GC/MS analysis as shown by the image below.

![Project](https://static.igem.org/mediawiki/2021/a/a2/T--WrightState--Results5.png)

The image above is a control sample containing α-terpineol spiked in (before extraction with ethyl acetate). A clear peak for α-terpineol can be seen at the timepoint of 18.40. The level of detection was determined by spiking in known amounts of α-terpineol in increasing concentrations and creating a calibration curve as shown below. Using this method, we were able to detect a spiked in concentration down to 10 - 15mM α-terpineol.

![Project](https://static.igem.org/mediawiki/2021/c/ce/T--WrightState--Results6.PNG)

Following the confirmation of the extraction method and level of detection, we were able to begin our production runs of α-terpineol. Sample data is shown below.

![Project](https://static.igem.org/mediawiki/2021/5/56/T--WrightState--Results7.png)

Above is an image from a production run of one of our samples which indicated no detection of α-terpineol. Based on the data above, it is inconclusive as to whether our system is producing α-terpineol. It is possible our system is producing α-terpineol at a level below the detection limit by GC/MS. Further tests and optimizations need to be conducted to clarify the results.

### Our address

**Wright State University**
**3640 Colonel Glenn Hwy.,
Dayton, OH 45435**
937-775-1000
Email Us

### Social media

@igemwrightstate