iGEM 2025 — Team rochester

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![Astronaut](https://static.igem.wiki/teams/5571/images/jessheaderstuff/notebook6.webp)
![DNA](https://static.igem.wiki/teams/5571/images/jessheaderstuff/dna.webp)

Notebook Entries
Protocols

# Brainstorming Period:

From the beginning of our collaboration as a team, we focused our efforts on conducting individual research to present ideas at collective brainstorming sessions. Regular meetings allowed for presenting and refining ideas, with regular peer and advisor feedback. Ideas were proposed and narrowed down until our current project was selected and team roles were assigned.

#### February 4th-Feburary 8th

* First official team meeting held.
* Brainstorm leader selected to assign research tasks and direct meeting trajectory.

#### February 9th- February 15th:

* Guidelines for team meetings established; presentation schedule organized.
* Presentations given by team members to propose project ideas.
* Workshop conducted by Wendy Way, STEM librarian at the University of Rochester on the following topics:

+ Compiling research
+ Creating visual guides
+ Analyzing primary literature

#### February 16th- February 22nd:

* Brainstorm efforts and presentations continued.
* Initial DBTL models created for project development.
* Team members continued to provide feedback on project ideas.

#### February 23rd- March 1st:

* Brainstorm efforts and presentations continued.
* Project ideas narrowed down to Top Ten proposals.
* Top Ten ideas formally presented to advisors for feedback.

#### March 2nd- March 8th:

* Team divided into groups to focus research efforts on the Top Ten. Research expanded to include:

+ Candidate genes, promoters, and host chassis
+ Local applications and intended impact
+ Early hardware and software concepts

* Group deliberations on Top Ten ideas continued; general presentations regarding project development created for advisor feedback.

#### March 9th- March 15th:

* Spring break: no team meetings held.
* Individual research and idea development continued.

#### March 16th- March 22nd:

* Group deliberations continued in preparation for “Top Three Ideas” meeting.
* Ideas narrowed down upon deliberation.

#### March 23rd- March 29th:

* Top Three Ideas meeting held; presentations given to advisors for feedback.
* Formal project deliberations held; final project selected.
* Leadership elections held; team roles finalized.

# Preparation and Protocol Development:

After finalizing the project idea, the team was organized into subcommittees for managing developing branches of the project as well as specific experimental modules, with a research focus on identifying key components. Experimental approaches were refined with advisor feedback and team workshops. Wet-lab protocols were developed, with associated training provided to prepare for experimental work.

#### March 30- April 5th:

* Subcommittees established:

+ Wet-Lab
+ Hardware
+ Modelling
+ Policy and Practice

* Project divided into 4 modules:

+ Autotrophic E. coli
+ Acetate Production
+ CRISPRi
+ PHB/PHV production

* Team divided into groups to identify BioBrick candidates for each module.

#### April 6th- April 12th:

* Research and refining ideas for BioBricks continued, with periodic advisor feedback* Ideas for hardware associated with each module conceptualized and refined* BioBrick workshop held, reviewing the following topics:
    + BioBrick components
    + Cloning methods
    + Insert verification

    #### April 13th- April 19th:

    * Project revisions continued.

    #### April 20th- April 26th:

    * Determination of assays for all modules began.

    #### April 27th- May 3rd

    * Assay protocols refined.
    * Lab safety protocols drafted following University of Rochester Environmental Health & Safety (EH&S) guidelines

    #### May 4th- May 10th

    * Final exam reading period: no team meetings held.

    #### May 11th- May 17th

    * Wet-lab bootcamp attended by all team members.
    + Basic lab techniques and safety were reviewed* Protocols developed for experiments to be run over the summer.
    * Potential donors and sponsors contacted.

# Wet Lab:

The team began the summer by culturing bacteria, preparing plasmids, and amplifying DNA. Established wet-lab protocols were refined and optimized for future experiments.

#### May 18th- May 31st

* First overnight cultures of DH5Alpha prepared.
* 40% Glycerol Bacterial Stocks prepared.
* BioBricks ordered.

#### June 1st- June 7th

* DH5Alpha and BL21 competent cells prepared.
* Plasmid backbones miniprepped: pSB1A3, pSB1C3, pSH4C5, pSB3T5, and pRHA-113
* Plasmids verified with restriction digest and gel electrophoresis.

#### June 8th- June 14th

* Stock of DH5Alpha and BL21 competent cells prepared and stored.
* Chemically competent cells transformed with miniprepped plasmids and plated on selection media.

#### June 15th- June 28th

* PCR amplification of BioBricks began.
* Transformations of 7 BioBrick parts into chemically competent cells performed.
* Optimization of PCR conditions on BioBrick DNA undertaken

+ Appropriate annealing temperatures troubleshooted
+ General primers used due to high specificity of amplification

#### June 29th- July 5th

* Optimization of colony PCR conditions undertaken.
* Diagnostic restriction digests using EcoRI and PstI performed to verify inserts
* Troubleshooting of 3T5 backbone cloning continued.

#### July 6th- July 12th

* PCR amplification for all parts completed and verified with gel electrophoresis.
* Ligation reactions and transformation of parts into DH5ɑ initiated.

#### July 13th- July 19th

* Efficiency of digestion and gel extraction optimized.
* 50μL PCR reactions for BioBrick template continued to obtain more DNA.

#### July 20th- July 26th

* High-fidelity restriction enzyme usage began to improve digestion efficiency.
* Primers specific to colony PCR verification designed.
* Workflow of digestion-ligation-transformation repeated.

#### July 27th- August 2nd

* Initial cloning efforts continued; number of transformants for cloned biobricks increased
* Troubleshooting of colony PCR continued; verification remained inconsistent.

#### August 3rd- August 23rd

* Cloned parts sent to PlasmidSaurus for sequencing.
* Attempts to identify and troubleshoot remaining colony PCR inefficiencies continued.

#### August 24th-August 30th

* pSB3T5 backbone replaced with pSB1C3 for improved results.
* Double digests on new constructs performed.
* Ligation and transformation into DH5ɑ E. coli cells conducted.

# Wrapping Up:

At the beginning of September, the team focused on verifying genetic constructs and performing associated assays. Data from assays, diagnostic digests, and growth experiments were collected and analyzed to prepare for documentation and uploading to the Wiki.

#### August 31st-September 6th

* Cloned parts sent to PlasmidSaurus for sequencing.
* Colony PCR continued.

+ Some growth of 1C3 parts verified.

* Diagnostic digests of BioBricks continued.
* Attempts to assemble and transform E. coli cells with BioBricks continued.

#### September 7th-September 20th

* Troubleshooting of colony PCR continued.
* Carbonic anhydrase and Bradford assays performed to test enzyme activity and protein concentration.
* Subcloning for assembling constructs continued.

#### September 21st-September 27th

* Protein assays and BioBrick transformation continued.
* Diagnostic digests for insert verification continued.

#### September 28th-October 4th

* Final diagnostic digests performed and gels ran for visualization of results.
* Assays for growth and acetate production continued.
* Growth curves for wild type E. coli strains grown in clinostat produced and analyzed.
* All assay data and gel images compiled in preparation for Wiki upload.
* Figure annotations and captions written in preparation for Wiki upload.

#### October 5th-October 8th

* Final assay data collection performed and uploaded in preparation for Wiki Freeze.

### Bacterial Culture

Lysogeny Broth (LB) Media Preparation

Cycle: Liquid autoclave · 90 min
Final volume: 1 L

#### Materials

|  |  |
| --- | --- |
| Bacto Tryptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 10 g |
| dH2O / ddH2O | to 1 L |

#### Equipment

* 2 L flask + magnetic stir bar
* 1 L graduated cylinder
* Autoclave machine
* Magnetic stir plate

#### Procedure

1. To a 2 L flask with a magnetic stir bar, add: 10 g Bacto Tryptone; 5 g Yeast Extract; 10 g NaCl.
2. Add ddH2O to a final volume of 1 L.
3. Mix well on a magnetic stir plate for 5–10 min until homogenous.
4. Autoclave for 90 min on liquid cycle.
5. After autoclaving, allow to cool to room temperature.

LB–Agar Plate Preparation

#### Materials

|  |  |
| --- | --- |
| Bacto Tryptone | 2 g |
| Yeast Extract | 1 g |
| NaCl | 2 g |
| Bacto Agar | 2.4 g |
| ddH2O | to 200 mL |
| Selection marker | per table |

#### Equipment

* 2 L flask + stir bar
* 1 L graduated cylinder
* Autoclave machine
* Bunsen burner
* Plastic bag + paper towel

#### Procedure

1. To a 2 L flask with a magnetic stir bar, add: 2 g Bacto Tryptone; 1 g Yeast Extract; 2 g NaCl; 2.4 g Bacto Agar.
2. Add ddH2O to a final volume of 200 mL.
3. Mix well 5–10 min until homogenous.
4. Autoclave for 30–60 min.
5. Allow to cool to ~60°C after autoclaving.
6. Add desired concentration of selection marker (see table).

#### Selection Marker Final Concentrations

| Selection Marker | Final Concentration |
| --- | --- |
| Ampicillin | 100 µg/mL |
| Tetracycline | 10 µg/mL |
| Chloramphenicol | 25 µg/mL |

7. Pour liquid mixture into plates next to a Bunsen burner and dry overnight.
8. Store plates at 4°C in a plastic bag with a paper towel to absorb moisture.

Liquid Bacterial Culture Preparation

#### Materials

* 5 mL Lysogeny Broth (LB)
* Antibiotic of choice
* Bacterial colonies

#### Equipment

* 10 mL culture (Falcon) tubes
* Gyrotory water bath shaker

#### Procedure

1. Add 5 mL LB to a culture tube.
2. Add desired antibiotic (see concentrations below).

#### Selection Marker Final Concentrations

| Selection Marker | Final Concentration |
| --- | --- |
| Ampicillin | 100 µg/mL |
| Tetracycline | 10 µg/mL |
| Chloramphenicol | 25 µg/mL |

3. Pick a single colony and inoculate the liquid.
4. Incubate at 37°C overnight with shaking at 200–250 rpm.

Competent E. coli Preparation (CaCl2)

#### Materials

* 100 mM CaCl2
* LB media
* Liquid nitrogen (for aliquot freezing)

#### Equipment

* Spectrophotometer
* 50 mL Falcon tubes
* High-speed centrifuge

#### Procedure

*Day 1*

1. Make 2 mL overnight liquid culture of *E. coli*.

*Day 2*

1. Add 1 mL of culture to LB medium to a final volume of 200 mL (target OD600 = 0.3–0.5) and split into four 50 mL aliquots.
2. Incubate on ice for 10 min.
3. Centrifuge at 4000 × g, 5 min, 4°C; discard supernatant.
4. Resuspend pellet in 25 mL 100 mM CaCl2.
5. Incubate on ice for 30 min.
6. Centrifuge at 4000 × g, 5 min, 4°C; discard supernatant. Resuspend pellets in 1 mL LB and combine cells in one tube.
7. Incubate on ice for 60 min.
8. Aliquot 100 µL; freeze in liquid nitrogen and store at −80°C until use.

Note: Preparing 100 mM CaCl2

11.1 g CaCl2 in 1 L H2O ≈ 0.1 mol·L−1 (Mr ≈ 110.99 g·mol−1).

M9 Minimal Media Preparation

#### Materials

* 12.8 g Na2HPO4·7H2O
* 3 g KH2PO4
* 0.5 g NaCl
* 2 g NH4Cl
* 1 mL 1 M MgSO4·7H2O (optional, post-autoclave)
* Carbon source (e.g., sugar or glycerol)
* Amino acids (if applicable; e.g., Threonine)

#### Equipment

* Erlenmeyer flask + stir bar
* Magnetic stir plate
* Autoclave

#### Procedure

1. In a flask with a magnetic stir bar, add salts (and amino acids if applicable).
2. Add distilled water to a final volume of 1 L.
3. Stir 5–10 min until homogeneous.
4. Autoclave 30–60 min.
5. Cool to room temperature.
6. Optional: add 1 mL 1 M MgSO4·7H2O.
7. Add carbon source (10 mL of 20% solution per chosen carbon); mix well.

Competent E. coli Transformation

#### Materials

* Competent cells
* Plasmid DNA (miniprep)
* LB media

#### Equipment

* 1.5 mL microcentrifuge tubes
* Gyrotory water bath shaker
* 37°C incubator / water bath, 42°C heat-shock bath

#### Procedure

1. Defrost competent cells on ice (do not warm quickly).
2. Add 50 µL competent cells to a pre-chilled 1.5 mL tube.
3. Add 1 µL plasmid to each tube. Negative control: add 1 µL LB.
4. Gently flick to mix (4–6 times); place on ice for 30 min.
5. Heat shock at 42°C for 30 s; return to ice 5 min.
6. Add 1 mL LB to each tube.
7. Recover 1 h at 37°C with gentle rocking.
8. Spin at 14,000 rpm for 30 s; remove all but 50–100 µL supernatant; resuspend pellet.
9. Plate on appropriate selective media.

Glycerol Stock Preparation

#### Materials

* 750 µL 40% glycerol
* 750 µL overnight culture
* 6 mL ddH2O (for 40% glycerol prep)
* Liquid nitrogen

#### Equipment

* 2 mL screw-top tube / cryovial
* Sterile inoculation loop

#### Procedure

1. Combine 750 µL overnight culture + 750 µL 40% glycerol in a cryovial; gently mix.
2. To make 40% glycerol: 4 mL 100% glycerol + 6 mL dH2O; autoclave.
3. Flash-freeze in liquid nitrogen; store at −80°C.

Note: Subsequent freeze–thaw cycles reduce shelf life.

#### Recovery

1. Using a sterile loop, scrape frozen material (do not thaw vial); streak on LB agar.
2. Grow overnight at the appropriate temperature.

E. coli Cryopreservation

#### Materials

* Liquid culture (high cell density)
* 1 mL 1× LB with 40% glycerol
* Liquid nitrogen (for flash-freezing)

#### Equipment

* High-speed centrifuge
* Sterile toothpicks

#### Procedure

1. Grow liquid culture in LB; confirm high density (e.g., OD600 for *E. coli*).
2. Pellet cells; resuspend in 1 mL 1× LB with 40% glycerol.
3. Flash-freeze; store at −80°C.
4. Streak cells with a sterile toothpick as needed.

### DNA Extraction and Isolation

Plasmid Miniprep (QIAprep® Spin Miniprep Kit)

#### Materials

|  |
| --- |
| 1–5 mL bacterial overnight culture |
| 250 µL Buffer P1 |
| 250 µL Buffer P2 |
| 250 µL Buffer N3 |
| 500 µL Buffer PB |
| 750 µL Buffer PE |
| 25 µL H2O |

#### Equipment

|  |
| --- |
| Tabletop & high-speed centrifuges |
| QIAprep 2.0 spin column |
| 1.5 mL microcentrifuge tubes |
| QIAGEN collection tubes |

#### Procedure

1. Pellet 1–5 mL overnight culture by centrifugation >8000 rpm (≈6800 × g) for 3 min at room temperature (15–25 °C).
2. Aspirate supernatant and resuspend pellet in 250 µL Buffer P1; transfer to a microcentrifuge tube.
3. Add 250 µL Buffer P2; invert 4–6× until clear (≤5 min).
4. Add 350 µL Buffer N3; invert 4–6×.
5. Centrifuge 10 min at 13,000 rpm (≈17,900 × g).
6. Load 800 µL supernatant onto a QIAprep column; spin 30–60 s; discard flow-through.
7. Wash with 500 µL Buffer PB; spin 30–60 s; discard.
8. Wash with 750 µL Buffer PE; spin 30–60 s; discard. Spin again 1 min to dry.
9. Place column in a clean 1.5 mL tube; add 25 µL nuclease-free H2O to membrane center; wait 1 min; spin 1 min to elute.

Polymerase Chain Reaction (PCR) — Q5 High-Fidelity

#### Materials

|  |
| --- |
| Q5 2X Master Mix (NEB) |
| Nuclease-free water |
| Forward & Reverse Primers |
| Template DNA |

#### Equipment

|  |
| --- |
| Benchtop centrifuge |
| Microcentrifuge tubes |
| Thermocycler |

#### Procedure

1. Set thermocycler to 98 °C.
2. Find Tm for each template.
3. Prepare PCR mix in a microcentrifuge tube.

| Component | 25 µL Reaction | Final Concentration |
| --- | --- | --- |
| Q5 High-Fidelity 2X Master Mix (NEB) | 12.5 µL | 1X |
| Forward Primer | 1.25 µL | 0.5 µM |
| Reverse Primer | 1.25 µL | 0.5 µM |
| Template DNA | 10 ng | — |
| Nuclease-free water | to 25 µL | — |

4. Aliquot 25 µL PCR MM into tubes.
5. Prepare a negative control (replace template with water).
6. Store PCR samples at 4 °C as needed.

#### Thermocycler Settings (25 µL Reaction)

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Initial Denaturation | 98 | 30 s |
| 35 Cycles | 98 | 10 s |
| 60 | 30 s |
| 72 | 90 s |
| Final Extension | 72 | 5 min |
| Hold | 4 | — |

*\* Extension times ~30 s/500 bp; optimize per insert.*

#### Post-PCR Checks

1. Run 0.8–1% agarose gel; visualize bands.
2. If single correct band: PCR cleanup → quantify.
3. If multiple bands: gel extract the correct band → quantify.
4. If no/faint band: try annealing gradient; repeat at 50 µL.

PCR Purification (QIAquick® PCR Purification Kit)

#### Materials

|  |
| --- |
| Buffer PB (with indicator) |
| 750 µL Buffer PE (ethanol-diluted) |
| 20–30 µL Buffer EB or nuclease-free water |
| Optional: 10 µL 3 M sodium acetate (pH 5.0) |

#### Equipment

|  |
| --- |
| Benchtop centrifuge |
| Microcentrifuge tubes |
| QIAquick spin columns |

#### Procedure

1. Add 5× Buffer PB to 1× PCR reaction; invert 4–6×.
   * If color turns orange/violet, add 10 µL 3 M sodium acetate (pH 5.0) — should return to yellow.
2. Load to column; spin 1 min; discard flow-through.
3. Wash with 750 µL Buffer PE; spin 1 min; discard.
4. Spin again 1 min to dry.
5. Elute with 20–30 µL EB/water (wait 1 min before spin).

Colony PCR

#### Materials

|  |
| --- |
| 2× DNA polymerase master mix (Taq) |
| 10 µM forward/reverse colony primers |
| Nuclease-free water |

#### Equipment

|  |
| --- |
| Benchtop centrifuge |
| Microcentrifuge & PCR tubes |
| Thermocycler |

#### Procedure

1. Master mix for 30 µL (per reaction):
   * 25 µL 2× polymerase master mix
   * 2.5 µL 10 µM forward primer
   * 2.5 µL 10 µM reverse primer
2. In tube: 30 µL nuclease-free water + 30 µL 2× mix (final 60 µL).
3. Touch a colony with sterile toothpick; swirl into tube.

#### Program

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Initial Denaturation | 98 | 30 s |
| 35 Cycles | 98 | 10 s |
| 60 | 30 s |
| 72 | 90 s |
| Final Extension | 72 | 5 min |
| Hold | 4 | ∞ |

Proceed to gel electrophoresis as needed.

Gel Electrophoresis

#### Materials

|  |
| --- |
| Agarose |
| ~50 mL 1× TAE Buffer |
| Sybr Safe (e.g., 1:10,000) |
| 1 kB DNA ladder |
| Parafilm |

#### Equipment

|  |
| --- |
| Erlenmeyer flask |
| Digital scale |
| Microwave |
| Electrophoresis chamber |
| Gel tray & comb |
| Gel documentation system |

#### Procedure

1. Weigh agarose per expected fragment size.

For 100 mL 1× TAE Buffer

| Agarose Mass (g) | % Agarose | Fragment Size |
| --- | --- | --- |
| 0.5 | 0.5 | 2 kb–50 kb |
| 0.8 | 0.8 | — |
| 1.0 | 1.0 | 400 bp–8 kb |
| 1.5 | 1.5 | 200 bp–3 kb |
| 2.0 | 2.0 | 100 bp–2 kb |

2. Microwave until dissolved; cool to 45–50 °C.
3. Add Sybr Safe (e.g., 5 µL/50 mL); swirl.
4. Pour gel; let solidify; remove comb and place gel in chamber.
5. Cover with 1× TAE. Load 1 kB ladder (2 µL).
6. Mix sample + loading dye on parafilm; load. Avoid bubbles.
7. Run 75–120 V until dye front ~80% down gel.
8. Image under UV/blue light.

### Cloning

Vector Restriction Digestion

#### Materials

|  |
| --- |
| Miniprep plasmid DNA |
| Nuclease-free water |
| 10× FastDigest Buffer |
| Restriction enzyme(s) |

#### Equipment

|  |
| --- |
| PCR tubes |

#### Procedure

1. Prepare a 20 µL digestion mix:
   * Calculate vector volume from concentration (e.g., to get 1 µg from 50 ng/µL, use 20 µL).
   * Add water to 20 µL total volume with 2 µL 10× buffer and 1 µL each enzyme.
2. Mix gently; quick spin.
3. Incubate 37 °C for 5–15 min; heat-inactivate if applicable.

Quick CIP Treatment (New England Biolabs)

#### Reaction Setup

Prepare a 20 µL reaction:

| Reagent | Amount |
| --- | --- |
| DNA | pmol DNA ends\* |
| rCutSmart™ 10× Buffer | 2 µL |
| Quick CIP | 1 µL |
| Nuclease-free water | to 20 µL\*\* |

\* ~1 µg of a 3 kb plasmid ≈ 1 pmol ends.

\*\* If enzymes cannot be heat-inactivated, purify DNA prior to ligation.

#### Procedure

1. Incubate 37 °C for 10 min.
2. Heat-inactivate 80 °C, 2 min.

Vector Ligation (T4 DNA Ligase)

#### Materials

|  |
| --- |
| Vector DNA |
| DNA insert |
| Nuclease-free H2O |
| T4 DNA Ligase |
| 10× T4 DNA Ligase Buffer |

#### Equipment

|  |
| --- |
| PCR tubes |
| Benchtop centrifuge |
| Thermocycler |

#### Procedure

1. For a 20 µL ligation (1:3 vector:insert molar ratio):
   * 2 µL 10× T4 Ligase Buffer\*
   * 50 ng vector (≈0.020 pmol)
   * 37.5 ng insert (≈0.060 pmol)
   * Nuclease-free H2O to 19 µL
   * 1 µL T4 DNA Ligase (add last)

\* Thaw/resuspend buffer at room temp (do not hand-warm).

2. Mix gently; quick spin (avoid bubbles).

Thermocycler Program — Cohesive (Sticky) Ends

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Cycle | 10 ↔ 30 | 30 s each · 71 cycles |
| Final | 65 | 10 min |
| Hold | 10 | ∞ |

3. Heat-inactivate at 65 °C for 10 min.
4. Chill on ice and transform 10 µL into 100 µL competent cells.

### Assays & Polymer Handling

Carbonic Anhydrase Assay

#### Method

The electrometric method of Wilbur and Anderson (1948) in which the time required (in seconds) for a saturated CO2 solution to lower the pH of 0.012 M Tris⋅HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T0; with enzyme, T.

#### Reagents

* 20 mM Tris⋅HCl buffer, pH 8.3. Store in an ice bath at 0-4°C before and during use.
* Carbon dioxide saturated water. Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay using sodastream (pressing button for 5 seconds for high carbonation). During saturation process, store water at 0-4°C in an ice bath.
* Enzyme

Add 1mL of cell lysate of E. coli with recombinant carbonic anhydrase to the initial volume of Tris.

#### Procedure

**Blank Determination:** Add 3.0 ml of chilled 20 mM Tris⋅HCl buffer, pH 8.3 to a 50 ml falcon tube in ice. Maintain temperature at 0-4°C and record pH.
Add 2 ml of chilled CO2 saturated water to the Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0.

**Enzyme Determination:** Add 2.0 ml of chilled 20 mM Tris⋅HCl buffer, pH 8.3 to a 50 ml falcon tube on ice. Maintain temperature at 0-4°C and record pH. Add 1 ml of enzyme sample and allow for pH to equilibrate. Quickly add 2 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.

#### Calculations:

<https://www.worthington-biochem.com/products/carbonic-anhydrase/assay>

Polymer Purification and Recovery Method

**MeCN (or DCM/THF) → Cold MeOH/EtOH**

**Lysate Cells and Weigh -**
Lyse cells (via sonication or an alternative method) and weigh 10 grams of the resulting cell lysate–Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granule mixture.

**Dissolve (5–10% w/v) -**
Add 100 mL acetonitrile to a beaker (for 10g PHBV)
Add 10 grams of lysate + plastic granule mix; stir at RT until fully dissolved (30–120 min).
Gentle warming to 30–35 °C can speed up; do not exceed solvent bp. Acetonitrile boils at 170ish, you can pump up the temp to much greater than 35 just a couple tens of degrees lower than the boiling point. keep in mind the melting point of pla as well, you don’t want to exceed that.

**Prepare Nonsolvent Bath -**
In a chilled beaker with a stir bar, add 500 mL of cold methanol. Place in an ice bath; aim for ~4–10 °C.

**Precipitate (Slow Addition) -**
With vigorous stirring, pour the PHBV solution slowly into the cold methanol (not the reverse). A white flocculent precipitate forms immediately. Rinse the solution vessel with a few mL of MeCN and add dropwise to maximize yield.

**Collect & Wash -** Vacuum filter through a Büchner funnel. Wash cake with 2× 50 mL cold MeOH to strip residual solvent.

**Dry -** Air-dry the cake in the hood 30–60 min, then vacuum-dry at 30–40 °C to constant mass. Gently crumble any soft agglomerates with a spatula to obtain free-flowing powder.

Source: team wiki notes. :contentReference[oaicite:0]{index=0}

One-Page SDS–PAGE + Fairbanks Staining

**1) Make 5% βME Laemmli buffer (per sample, 20 µL)**
10 µL 2× Laemmli (no βME)
9 µL H₂O
1 µL β-mercaptoethanol (βME) → 5% v/v
For n samples, make (n+1)× master mix (20 µL per sample).

**2) Denature samples (final 1× Laemmli)**
Thaw samples briefly at 23 °C, then place on ice.
Mix 20 µL 5% βME Laemmli + 20 µL protein sample (lysate supernatant).
Cap tightly; quick vortex + spin.
Heat 95 °C, 10 min.
Spin 1000 g, 5 min. Keep on ice.

**3) Set up gel/tank (4–20% SDS–PAGE)**
Remove bottom tape; place gel in holder.
If single gel, insert back plate.
Fill inner chamber with 1× TGS; fill outer tank to ~¼ height.
Remove comb straight up.
Rinse wells with ~15 µL 1× TGS each.

**4) Load & run**
Load 5 µL prestained ladder.
Load 10 µL denatured supernatant per sample.
Run 100 V, 10 min (stacking).
Then 130 V until dye front ~⅔ down gel (~50–60 min).
Do not run off the gel.

**5) Retrieve gel**
Open cassette; transfer gel to a staining container.

**6) Fairbanks staining (A → B → C → D; 100–150 mL each)**
For each solution (A, then B, then C, then D):
Submerge gel in 100–150 mL solution.
Microwave 80–90 s until just boiling.
Immediately move to fume hood; rock gently 1 min to cool.
Drain and rinse gel with ddH₂O.

**7) Image**
Place gel on white screen/plate in the imager.
Select Coomassie Blue program; adjust sliders for clarity.
Save images (e.g., PNG/JPG) and archive a .tif copy.

**Fairbanks recipes (1 L each)**
A: 0.05% Coomassie (50 mL of 1%), 25% isopropanol (250 mL), 10% acetic acid (100 mL), ddH₂O to 1 L (~600 mL).
B: 0.005% Coomassie (5 mL of 1%), 10% isopropanol (100 mL), 10% acetic acid (100 mL), ddH₂O to 1 L (~795 mL).
C: 0.002% Coomassie (2 mL of 1%), 10% acetic acid (100 mL), ddH₂O to 1 L (~898 mL).
D: 10% acetic acid (100 mL), ddH₂O to 1 L (~900 mL).

Source: team wiki notes. :contentReference[oaicite:1]{index=1}

Coomassie Plus (Bradford) Protein Assay – Quick Protocol

**A) Prep: Reagent & Standards**

*Equilibrate & mix reagent*
Gently invert the Coomassie Plus (Bradford) Assay Reagent a few times (do not shake). Bring to room temp before use.

*Prepare protein standards (BSA recommended)*
Use BSA 2.0 mg/mL stock to make a standard curve spanning your working range.

*Standard working ranges:*
Plate / test-tube (standard): 100–1500 μg/mL (linear with BSA ~125–1000 μg/mL).
Low-range (micro format): 1–25 μg/mL.

Example dilutions are provided below (use the same diluent as your samples).

*Example BSA standards (standard range, for plate or test tube)*
2000, 1500, 1000, 750, 500, 250, 125, 25, 0 μg/mL

*Example BSA standards (low-range, for micro format)*
25, 20, 15, 10, 5, 2.5, 0 μg/mL

Add 50 μL standard or sample to labeled tubes.
Add 1.5 mL Bradford reagent, mix well.
Zero spectrophotometer with water; read A595.
Blank-correct and quantify from the standard curve (linear range with BSA ~125–1000 μg/mL).

ThermoFisher user guide (for reference):
[MAN0011344](https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011344_CoomassiePlus_Bradford_Asy_Reag_UG.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block body\_class %}engineering-page{% endblock %}
{% block page\_content %}

![Astronaut](https://static.igem.wiki/teams/5571/images/jessheaderstuff/engin6.webp)
![DNA](https://static.igem.wiki/teams/5571/images/jessheaderstuff/dna.webp)

* [Overview](#eng-overview)
* [Spinova Clinostat](#spinova)
  + [Design](#sp-design)
  + [Build](#sp-build)
  + [Test](#sp-test)
  + [Iteration](#sp-iteration)
  + [Learn](#sp-learn)
* [Filament Extruder](#extruder)
  + [Overview](#ex-overview)
  + [Casing (V1–V4)](#ex-design)
  + [Filament Winder](#ex-build)
  + [Plastic Input](#ex-test)
* [References](#eng-refs)

## Overview

To sustain human extraterrestrial presence, biologists and engineers must overcome the challenges presented by extreme temperatures, limited resources, and unique environmental conditions. These variables impact the capability of biomanufacturing which enables prolonged space exploration. To address these challenges, our team employs PHAntom Spinova: a two-axis clinostat machine that continuously reorients a sample to mimic the effects of microgravity. Spinova enables our team to model bacterial behavior in simulated microgravity conditions without leaving Earth’s atmosphere.

Bioplastics such as Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), produced by bacterial synthesis, offer a biodegradable and renewable solution, but converting these raw materials into usable forms remains a challenge. To bridge this gap, our team developed the PHBV Filament Extruder: a device that uptakes PHBV granules and processes them into uniform, high-quality 3D printing filament. By directly linking synthetic bioplastic production with filament production, the extruder enables an efficient pathway from sustainable material creation to practical use via 3D printing.

## Spinova (Two-Axis Clinostat)

Engineering Design Process (Spinova)

Spinova was designed using the cloud-based CAD software OnShape, and printed using PLA filament on an Ultimaker 3 Fused Deposition Modeling (FDM) printer using Ultimaker Cura 3D slicing software. Our design took inspiration from CoreElectronics’ Random Positioning Machine, and we started with a top-down approach to parts. In the end, Spinova went through over 5 different iterations and is still accepting and implementing user feedback!

* Drafts of parts are dependent on relevant dimensions and locations of other sections of the design, meaning a single sketch or feature can drive the geometry of several parts in the overall design.
* Each solid part of the clinostat was modeled in its own independent parts studio in OnShape, then combined in an assembly.
* We followed an iterative design process where surveys were sent out to potential end-users and to those knowledgeable about mechanical engineering and design.
* The surveys asked for feedback on each iteration of Spinova, which was then revised in-house and updated following the feedback we received.

### Design

The primary software used to design the hardware was OnShape, and gears were designed using the gear generator from STLGears.com. The design for Spinova began as a two-dimensional sketch of the individual rotating frames and the sample platform. As the frames were designed, dimensions to keep them from interfering with each other were added.

Once the initial frame dimensions were completed, we extruded the 2D sketches into 3D shapes. At this point, each individual part was given its own parts studio in OnShape so they could be worked on independently.

At this point in the design, we began to reach out to potential end-users for feedback on our preliminary design. We received feedback that was implemented into the subsequent designs for the clinostat. More information about our feedback process can be found below. Our initial design was a three-axis clinostat, however for practicality, space and weight considerations, and based on user feedback the design was updated to be a two-axis clinostat.

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›

![Top-down concept and frames](https://static.igem.wiki/teams/5571/images/engineering2/s1.webp)

Top view of the preliminary geometry. Dotted construction lines are for reference; solid closed profiles define extrusions.

![Dimensioned 2D layout](https://static.igem.wiki/teams/5571/images/engineering2/s2.webp)

Dimensioned 2D layout of the sample platform, inner frame, and outer frame—clearances prevent interference.

![Early platform](https://static.igem.wiki/teams/5571/images/engineering2/s3.webp)

Early experiment platform with printed through-axles and no O-ring groove yet.

![Early inner frame](https://static.igem.wiki/teams/5571/images/engineering2/s4.webp)

Early inner rotating frame—minimal ring with integral pegs, lacking motor mounts and bearing seats.

![Outer frame later iteration](https://static.igem.wiki/teams/5571/images/engineering2/s5.webp)

Later stationary outer frame iteration with corner cut-outs for legs and a stiffened rim.

### Build

Once components were designed and ready to be 3D printed, they were uploaded to the 3D printing software UltiMaker Cura for slicing. 3D printed components apart from the table legs were joined using press fit tolerances between steel dowel pins and roller bearings.

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›

![Three-axis concept](https://static.igem.wiki/teams/5571/images/engineering2/s8.webp)

Three-axis clinostat concept explored during ideation—ultimately dropped for mass/space/complexity.

![Preliminary assembly for feedback](https://static.igem.wiki/teams/5571/images/engineering2/s7.webp)

Preliminary Spinova assembly that received the first round of user feedback, responses allowed us to focus our design on a two-axis model.

### Test

Precision and Stability (RPM consistency check)

To validate the design decisions we made to follow our specified requirements, we conducted a series of functionality tests with the goal of correlating our design requirements to Spinova’s real-life specifications.

**Purpose:** ensure 3.4:3.8 RPM rotation ratio is maintained for consistent simulation of Martian gravity conditions.

**Method:** Use a tachometer to assess rotation speed of the sample platform and the inner rotating frame.

**Criteria for success:** Average speed for sample platform 3.4 +/- .1 RPM, avg speed for inner frame 3.8 +/- .1 RPM, stable over time with >4% variance. By setting a 4% variance limit, we accept a small performance deviation that is within a biologically acceptable range for E. *coli.* Further testing is an option to validate that E. *coli* remains biologically stable through growth curves and phenotype monitoring. To have the inner rotating frame rotate at 3.4 RPM, the motor must rotate at 11.6 RPM. To have the sample platform rotate at 3.8 RPM, the motor must rotate at 13.4 RPM. These calculated RPM for the motor come from the gear ratio and speed relationship.

* Gear ratio = Driven teeth/ Driver teeth
* Driven speed = Driver speed x (Driver teeth / Driven teeth)
* For the rotation of the inner rotating frame: Driver speed = 3.4 RPM x (75/22) → 11.6 RPM
* For the rotation of the sample platform: Driver speed = 3.8 RPM x (60/17) → 13.4 RPM

**Relevant design choices:** Selected the FS90R continuous microservo motor with a range of rotation speeds and used gears to mediate transfer of torque.

**Results:** When running Spinova with liquid cultures of modified BL21, laser tachometer readings indicated consistent RPM at each 30-minute interval tested. The recorded speed of the inner rotating frame was 11.5 RPM for each time interval and the recorded speed of the sample platform was 13.5 RPM for each time interval. This is within our set tolerance limits.

Portable Design (weight, volume, thermal analysis)

**Purpose:** Ensure Spinova meets physical constraints imposed for lab/shuttle use.

**Method:** Measure weight of all components in CAD and in print to check for deviations in weight, measure actual dimensions to assess deviations in size, place in incubator at 37 C to assess thermal tolerance.

**Criteria for Success:** Must weigh under 0.375 kg with less than a 10% error margin, fit on a standard lab bench, and maintain structural integrity at biological sample culture temps 37 C. ESA technical constraints for drop tower experiments, which is a relatively small hardware component for an experiment, outlines mass and space allocations for small projects in the setting of a space mission (ESA). These constraints guided our goal weight for Spinova.

**Relevant design choices:** Used PLA plastic to 3D print plastic components and designed dimensions to fit on a laboratory bench while in use.

**Results:** Model weight was calculated in OnShape using the “assign material” feature and found to weigh 0.371 Kg, including estimated weight for non-3D printed materials. Actual weight was calculated with a lab scale and found to weigh 0.338 Kg. This resulted in a 9.87% error between the CAD and the physical model. The source of this error was likely due to the CAD model weight not accounting for the infill the print was actually run with. All Spinova 3D printed components were printed at 15% infill. The actual dimensions of Spinova were measured to be 251.00 × 251.00 × 149.48 mm. To test the thermal tolerance of Spinova, it was run continuously for two hours in a 37 C incubator without a sample. Periodic checks revealed no structural deformation. Subsequent wet lab experiments were run with Spinova and found metabolic processes remained optimal and intact when modified BL21 were cultured overnight in Spinova. A graph overlaid with non-Spinova control cultures is displayed.

![Growth curve comparison](https://static.igem.wiki/teams/5571/images/engineering2/s9.webp)

Growth curve comparison: cultures on Spinova (simulated microgravity) outperform non-shaking controls and track above shaking controls.

Simple Assembly

**Purpose:** Ensure Spinova can be assembled in a timely manner without specialized knowledge or tools that are not already carried by astronauts for routine spacecraft maintenance.

**Method:** Time assembly of Spinova, then time assembly of Spinova for someone who did not design it. Assess if tools used for assembly deviate from those described in the user manual.

**Criteria for success:** Assembly under 5 minutes, only using user manual instructions and tools.

**Relevant design choices:** Some parts like the table legs are attached using super glue and don’t need to be installed every use. Tolerances were employed to allow close fits while maintaining freedom of rotation where appropriate.

**Results:** Spinova assembly time was recorded for various lab members, as well as individuals unaffiliated with the iGEM team. Subjects were given a small Philips head screwdriver, zip ties, and rubber hammer to assemble Spinova using the instructions provided in the user manual. An average assembly time of 270 seconds (4 minutes and 30 seconds) was recorded among seven participants. Note, time does not include time to adhere table legs to the outer stationary frame.

![Assembly time trials](https://static.igem.wiki/teams/5571/images/engineering2/s10.webp)

Assembly time trials: average 270 s (4:30)—comfortably under the 5-minute target.

### Iteration

Concepts for the initial design included a version that was supported by two stands. However, based on feedback from our advisors and wet lab team, we opted for a table top design with four leg supports.

In previous Spinova design concepts, the axles connecting the sample platform to the inner frame, and the ones connecting the inner frame to the stationary frame were to be one print with the component they supported. After surveying students and staff in both the biomedical engineering and mechanical engineering departments of the University of Rochester, Spinova evolved again to use steel dowel pins to connect the frames. The use of dowel pins inserted in axial ball bearings as shafts allowed for more simple assembly and manufacturing, as one shaft could be installed at a time, and we were able to alter the dimensions in OnShape for a metal to plastic press fit between the shafts and the receiving holes.

Even after achieving a physical print of Spinova, iteration and design changes did not end. Post processing played a significant role in a functional clinostat. Parts that seemed to work out mathematically and on the computer model were not always a perfect representation of real life. 3D printers have some degree of error that can be mostly mitigated by an excellent design. Our solution was to work with specific tolerances for our 3D printed parts. For press fits, a tolerance of 0.25 mm was standard across our design. While striving to achieve that ideal design, the original Spinova prints were altered using various hand tools in order to get a functioning model.

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![Two-stand concept from literature](https://static.igem.wiki/teams/5571/images/engineering2/s11.webp)

Literature concept using two external support stands (Vashi et al., 2022) that informed early thinking.

![Four-legged tabletop design from literature](https://static.igem.wiki/teams/5571/images/engineering2/s12.webp)

Four-legged tabletop clinostat (Cacayurin, 2023) that influenced our legged design after feedback round 2.

![Hand finishing for tolerance](https://static.igem.wiki/teams/5571/images/engineering2/s13.webp)

Hand-finishing: widening the stationary-frame hole with a Dremel to ensure free slip-ring rotation.

### Learn

In the spirit of the iterative design-build-test-learn cycle, we implemented feedback from students, faculty, and potential end users of our technology which ultimately helped shape Spinova’s current design. We asked what features potential end-users would like to see, and in what applications they could use our hardware. Feedback was then sorted and implemented as it was received, meaning Spinova was constantly evolving.

**Design changes directly resulting from the feedback forms include:**

* Groove in the sample platform for an O-ring to hold the sample with friction
* Switching from 3D printed axles to steel dowel pin shafts with corresponding holes in the frames
* Addition of supports to the legs of the outer stationary frame
* Ball bearings to allow smooth rotation of the frames

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›

![Spinova in operation](https://static.igem.wiki/teams/5571/images/engineering2/s14.webp)

Hardware Feedback Form

![Feedback form UI](https://static.igem.wiki/teams/5571/images/engineering2/s15.webp)

Hardware Feedback Form Responses

![Feedback-to-resolution table](https://static.igem.wiki/teams/5571/images/engineering2/s16.webp)

Compiled feedback with corresponding design resolutions (bearings, O-ring groove, dowel pins).

![O-ring groove implemented](https://static.igem.wiki/teams/5571/images/engineering2/s17.webp)

Updated experiment platform showing the added O-ring groove for frictional sample retention.

## Extruder Hardware

### Filanova Extruder

This hardware component was created as a potential method to process the plastic produced by our E. *coli* into printable filament. The objective for the extruder was to create a low-cost, versatile system capable of processing various plastic materials, including old ground-up filament, standard plastic pellets, and most importantly, the novel cell-produced PHA from our bacteria, into consistent 1.75 mm filament for 3D printing.

The extruder was inspired by the V5 filament recycler created by Lyman and Mullier on Thingiverse, with modifications for cost reduction and compatibility with native Ender3 printer components. The functional design required a way for plastic pellets to be melted and pushed through a die to form filament that could be used for 3D printing. The melting chamber of the extruder consisted of a ½ inch ID steel pipe, selected for availability reasons and fit for a ⅝ inch auger to rotate freely inside. This pipe was heated by a 1-inch band heater, operating in a testing temperature range of 200 to 230 °C, and was fitted with a brass plug nozzle to form the final 1.75 mm extrusion die. These would be screwed to a metal flange on the main casing with a block of pine wood sandwiched in between to prevent heat creep from warping the printed casing. A high-torque Nema 23 stepper motor was chosen over a Nema 17 to provide the necessary force to push both solid and melted plastic. This motor drove a standard 5/8-inch auger, which was modified by cutting 92.5 mm off its tip to fit the assembly.

The filament winder was based on a design by shoaibkhan786 for a PET bottle recycler on Thingiverse. Its purpose was to provide a slow rotation for a collection of the extruded filament into a reel. It was powered by a NEMA 17 motor, with speed reduced through a system of five 3D-printed gears.

Design tools included Onshape for precision parts and TinkerCAD for assemblies. All parts were sliced using Ultimaker Cura and printed on an Ender3 FDM printer using PLA plastic. Printed supports and other waste were blended into small pieces and used as testing material for the final extruder.

### Extruder Casing

Version 1

The design for the extruder casing was created with a combination of Onshape and TinkerCAD. The casing was modeled around the dimensions to fit the functional parts of the extruder. Parts were sliced with Ultimaker Cura and printed using an Ender3 FDM printer with PLA.

The extruder base was designed as a single piece to hold together the main components of the extruder. The base, in particular, had to be able to hold an auger attached to a Nema 23 motor at a 90 degree angle from the base, so that the auger could go through the metal pipes that fed into the hot end without jamming or getting stuck. An inverted pyramid created a feeding tray for plastic to be fed into the pipe by the rotating auger.

The design of this base was both too large and too inconvenient, both in wasted filament on supports and on time, to be printed on the lab’s Ender3, as it simply did not fit on the print bed. A decision was made to break it up into modular pieces that could be snapped together like legos and redesigned independently.

The design was broken down into nine separate pieces that would be designed to snap together. The largest and most complex component, the collection tray, was printed as a single part. To determine the optimal connection method, we conducted small-scale printing tests comparing pegs with slot-based fittings. The slot design failed due to printing inaccuracies from large overhangs, resulting in poor fits. In contrast, the peg design was easier to print reliably and allowed for simple post-processing. When the printer's offset—a difference of around 3 mm between digital and physical parts—caused fit issues, the pegs could be easily shaped with a Dremel tool to ensure a proper snap-in connection.

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![Modular casing with pegs](https://static.igem.wiki/teams/5571/images/engineering2/e2.webp)

Modular casing with snap-fit pegs added across frames.

![Peg vs slot test prints](https://static.igem.wiki/teams/5571/images/engineering2/e8.webp)

Small test prints comparing peg vs. slot connections—pegs printed reliably and were easy to tune with a Dremel.

Version 2

The second extruder housing was modular and could be snapped together using the peg connections. The separated parts could be individually modified to account for errors during the design process. These parts were printed in separate batches with PLA filament using an Ender3 printer with 20% infill.

Parts were tested for fit after printing. Measurement errors, such as misplaced pilot holes for screws on the plate holding the metal flange, were fixed immediately after.

Fit testing with all our printed parts showed that the front plate with the hot end of the extruder had difficulty snapping onto the rest of the casing. The screw heads on the other side of the flanges were running into the plastic of the collection tray, preventing it from snapping all the way. Additionally, the body housing the auger to motor coupler was too short to fully house the shank of the auger. The plate attaching the NEMA 23 was also misaligned in its screw holes. We learned from this version that our previous measurements, taken from online documentation, were not precise enough for our purposes. We manually took measurements from our ordered parts using calipers and rulers for designing the next iteration.

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![Front plate and pegs around hot-end](https://static.igem.wiki/teams/5571/images/engineering2/e3.webp)

Front-plate and peg placements around the hot-end opening.

![Motor/flange plates, hole updates](https://static.igem.wiki/teams/5571/images/engineering2/e4.webp)

Motor/flange plates with hole locations updated from vendor drawings to caliper measurements.

Version 3

Version 3 of the extruder casing used the improved measurement information from V2. It had sharper angles for the collection tray to move the walls out of the way of the screws. The sharper angle also helped in causing plastic in the tray to fall and be guided towards the rotating auger. An additional section of the casing housing the auger was added to allow for the length of the auger and to further stabilize it. The plate holding the NEMA 23 was also redesigned in accordance with the physical screw placement of the motor.

Version 3 was assembled and tested following the complete assembly of the extruder. It was found that the weight of the auger caused the angle to be off from 90 degrees, where it was tilting down and pressing against the tube lightly. Due to this, when the motor was turned on, the front half the casing would lightly oscillate from the movement of the motor. The weight of the nema was also an issue since the extruder would frequently buck during oscillations and fall over from the housing collapsing due to the removable pegs. Additionally, the front plate holding the extruder did not snap in tightly due to the fact that screwing the plate to the flange was mildly warping the plastic, causing it to not be completely straight. This led to large gaps through which plastic could fall out of and a collection tray that would easily fall apart with movement. The modularity and pegs themselves also became issues as they were too loose to hold together the entire extruder. We learned that the modular, snap-fit design was not suited for the mechanical load, and the system needed to be anchored to a rigid external frame.

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›

![Collection tray angle redesign](https://static.igem.wiki/teams/5571/images/engineering2/e6.webp)

Collection-tray redesign with steeper angle to guide pellets and clear hardware.

![V3 printed assembly bench test](https://static.igem.wiki/teams/5571/images/engineering2/e5.webp)

V3 printed assembly during bench test—oscillation/buckling issues observed.

Version 4

Through testing Version 3, we determined that a fully 3D-printed housing for the filament extruder was not sturdy enough. We changed to eliminating the housing for the motor and everything except the collection tray. The collection tray was also redesigned to print in a single part, eliminating the use for pegs, to eliminate any gaps and better hold the tray together under strain. A base for the tray was also printed with holes that would fit the legs of the collection tray, so it can be easily removed, and had screw holes that could anchor it down to a base. Metal bushings were added to the holes that the auger would pass through to eliminate the auger cutting into plastic and bucking around with the purchase. This helped stabilize the movement of the auger. The housing was replaced with a base made of 2’’ thick pine wood, cut with a hacksaw, both to pad against the heat generated by the extruder and to create a solid surface it could be anchored onto. Blocks of wood were screwed onto the wooden base with metal brackets to brace the extruder against horizontal movement and to keep it easily removable for fixes. This version was tested and it successfully extruded filament without human intervention, as we did not have to hold together the extruder parts to prevent them from moving.

![Hot-end detail](https://static.igem.wiki/teams/5571/images/engineering2/e7.webp)

Hot-end detail: ½″ pipe, 1″ band heater, 1.75 mm brass die.

### Filament Winder

Version 1

The filament winder was based off a design from shoaibkhan786 for a PET bottle recycler on Thingiverse. The gears were modeled in Onshape and the rest was modeled in TinkerCAD. The winder is powered off a NEMA17 motor and slowed down through 5 gears to slowly turn a collection reel. Parts were printed in batches and in PLA plastic. 6 19 mm OD 7 mm ID ball bearings were inserted into each side (3 per side) with 7 mm printed rods going through the center to allow for free rotation of the gears on the rods. Gears were superglued down on the rods to prevent movement and shifting. The NEMA 17 is mounted to the side with a block placed under to better distribute the weight and hold up the motor. 4 mm 65 mm screws and nuts were inserted in each corner and the bottom block to hold together the winder. Version 1 was tested and to have been found functional with no major flaws, confirming that the gear train was able to rotate the collection reel at a much slower rate successfully from the NEMA 17 motor.

### Plastic Input

Version 1

To test the initial performance of the extruder, we used PLA filament readily available in the lab. We chopped the PLA filament reel into small pieces to accurately mimic the size of PHA granules typically extracted from microbes. This input allowed us to successfully produce filament using the extruder; however, the auger drill occasionally became stuck when unevenly cut pieces lodged between the drill and the hot end.

Version 2

To prevent the auger drill from getting stuck, we decided to use a more powder-like material resembling the plastic granules expected after microbial extraction and purification. For this, we used waste or byproducts from previous 3D printing projects and blended them into a fine powder. We then took this a step further by applying a polymer purification and recovery method (can find the exact procedure in our Wet Lab Protocol section) to purify and precipitate plastics such as PLA and PHA, forming a soft, fluffy, and airy substance that extrudes more efficiently, with minimal interference between the drill and the hot end.

References

* Lyman / Mulier Filament Extruder V5 by hlyman - thingiverse. (n.d.-a).
  [https://www.thingiverse.com/thing:380987](https://www.thingiverse.com/thing%3A380987)
* Pet plastic filament recycler by shoaibkhan786 - thingiverse. (n.d.-b).
  [https://www.thingiverse.com/thing:6215434](https://www.thingiverse.com/thing%3A6215434)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}
Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate your work.
{% endblock %}
{% block page\_content %}

![Astronaut](https://static.igem.wiki/teams/5571/images/jessheaderstuff/experiments5.webp)
![DNA](https://static.igem.wiki/teams/5571/images/jessheaderstuff/dna.webp)

* [Strain 1](#strain1)
  + [Core Design](#s1-core-design)
  + [Autotrophy module](#s1-autotrophy)
  + [Part characterization](#s1-part-char)
  + [Final transformation](#s1-final-xform)
* [Strain 2](#strain2)
  + [Core Design](#s2-core-design)
  + [PHBV module](#s2-phbv-module)
  + [Part characterization](#s2-parts)
  + [CRISPRi module](#crispri-module)
  + [Final transformation](#s2-final-xform)
* [Protein Characterization](#PROTCHAR)
  + [T7-CRP msGFP2](#T7MSGFP2)
  + [T7-lacO rhc\_cbbL](#T7RHC)
  + [saz\_αCA–4C5](#T7SAZ)
* [References](#references)
  + [All references](#references)

## Strain 1: Autotrophic Acetate Producer

### Core Design

#### Goals

* Enable *E. coli* autotrophy via a minimal CBB cycle.
* Overproduce & secrete acetate (pta–ackA).

Autotrophy module (pCBB⁺ + FDH helper)

The **Autotrophic Acetate Producer Module** is designed as the upstream half of our two-strain system, supplying a stable and storable carbon intermediate to the PHBV production chassis. Instead of relying on a natural acetogen (such as *S. ovata*), we proposed engineering an **autotrophic *E. coli*** to couple **CO2 fixation** with **acetate secretion**.

#### Core Design

1. **Autotrophy**

   We engineer ***E. coli* BL21(DE3)** for autotrophic growth by installing a minimal Calvin-cycle module (cf. Nissan et al., 2024) plus CO2-supply and for enhanced reduction supports. The goal was to enable *E. coli* to **fix atmospheric CO2 as its primary carbon source**, reducing dependence on sugars and enabling sustainable biomanufacturing in resource-limited settings.
2. **Acetate Production**

   We engineer ***E. coli* BL21(DE3)** for the overproduction and secretion of acetate through expression of a recombinant pta–ackA pathway.

#### Construction of our Autotrophy Expression Module

Our autotrophy module expresses **cbbL/cbbS** (Form I RuBisCO, *R. capsulatus*), **rbcX** (*Picosynechococcus sp.*), and **prkA** (*R. sphaeroides*), with **FDH** (*Pseudomonas sp. 101*) for enhanced reduction and **β-CA** (*Dolichospermum circinale*) to enhance local CO2 near RuBisCO by dehydrating local HCO3- to CO2.

These genes are expressed using a two-plasmid system: the megaplasmid **pCBB+**, encoding the minimal Calvin–Benson–Bassham (CBB) module plus the supporting β-carbonic anhydrase (β-CA), and a companion plasmid expressing **formate dehydrogenase (FDH)**.

1. The pCBB⁺ megaplasmid was assembled in pSB1C3— a high-copy CmR vector with a ColE1/pMB1-derived ori—by cloning two composite, T7–lacO–driven operons. Two polycistronic cassettes—(i) `rhc_cbbL–psp_rbcX–rhc_cbbS` and (ii) `rhs_prkA–dci_bCA`—are each driven by a T7–lacO regulatory region (T7 promoter with a lac operator for IPTG-tunable, T7 RNAP–dependent expression). Each cassette includes a ribosome binding site (RBS, BBa\_B0034) upstream of every coding sequence (CDS) and terminates with the iGEM double terminator BBa\_B0015 (rrnB T1 + T7Te).
2. The FDH plasmid was assembled in pSB3T5 (BioBrick prefix/suffix), a medium-copy TetR vector with a p15A origin. The insert encodes `psp_FDH` (*Pseudomonas sp. 101*) under the lac promoter, with a strong RBS (BBa\_B0034) immediately upstream of the coding sequence, and with transcription terminated by the iGEM double terminator BBa\_B0015 (rrnB T1 + T7Te).

### Individual Part Characterization & Data

Individual parts (cbbL, rbcX/cbbS, prkA)

#### rhc\_cbbL — *Rhodobacter capsulatus* Form I RuBisCO large subunit (UniProt: O32740 · RBL1\_RHOCB)

The *Rhodobacter capsulatus* `cbbL` gene encodes the large subunit of Form I RuBisCO.
In Form I RuBisCO, eight large subunits assemble with eight small subunits (`rhc_cbbS`) to form the
L8S8 holoenzyme. The large subunit houses the active site which catalyzes the carboxylation
of ribulose-1,5-bisphosphate (RuBP) to yield two molecules of 3-phosphoglycerate
(RuBP + CO2 + H2O → 2 × 3-PGA). Like all RuBisCOs, Form I enzymes can also catalyze an
oxygenase side reaction (RuBP + O2 → 3-PGA + 2-phosphoglycolate), establishing a trade-off between
CO2 fixation and photorespiratory flux (Andersson, 2007).

In engineered systems, `cbbL` can be recombinantly co-expressed with `cbbS` (and typically
`prkA`, phosphoribulokinase) to reconstitute the Calvin–Benson–Bassham (CBB) cycle in heterotrophs
like *E. coli* (Nissan et al., 2024). Optimal performance generally requires chaperone-assisted
folding/assembly (e.g., GroEL/ES and/or RbcX) and a balanced RuBP supply to favor the carboxylase reaction
(Nissan et al., 2024).

In our system, we co-expressed `rhc_cbbL`/`rhc_cbbS` and `prkA` with a
β-carbonic anhydrase from *Dolichospermum circinale* based on these considerations for optimal
performance. These enzymes route fixed carbon through glycolysis to acetyl-CoA, which feeds our PHB/V
biosynthetic pathway.

##### BioBrick

The `rhc_cbbL` coding sequence (UniProt O32740, RBL1\_RHOCB; *R. capsulatus* Form I RuBisCO
large subunit) was assembled as a BioBrick and ordered from IDT. The `rhc_cbbL` BioBrick is flanked
by the standard prefix/suffix, and expressed from a T7–lac cassette (T7 promoter with a lac operator for
IPTG-tunable control). An RBS (BBa\_B0034) is positioned immediately upstream of the coding region within the
cassette.

![rhc_cbbL schematic](https://static.igem.wiki/teams/5571/images/danielwet/1.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![rhc_cbbL PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/screenshot-2025-10-07-194933.webp)

Successful PCR of the **rhc\_cbbL BioBrick** with general F/R primers, showing the expected ~1.6 kb band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `rhc_cbbL` insert was cloned into the iGEM backbone pSB1C3. The PCR product (`rhc_cbbL`)
and pSB1C3 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR & DNA
Cleanup Kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector
were ligated in a 3:1 ratio to generate pSB1C3::rhc\_cbbL, transformed into *E. coli*, selected on chloramphenicol
plates, and verified by colony PCR and EcoRI/PstI diagnostic digest. Final candidate clones were submitted to
Plasmidsaurus for sequencing.

#### psp\_rbcX / rhc\_cbbS bicistronic unit — *Picosynechococcus* RuBisCO chaperone RbcX (UniProt: Q44177 · RBCX\_PICP2) & *R. capsulatus* RuBisCO small subunit (UniProt: O32741)

RuBisCO chaperone (`rbcX`) is a specific assembly chaperone that acts downstream of GroEL/ES to
promote formation of the RuBisCO large-subunit core (Saschenbrecker et al., 2007). As a homodimer
(RbcX2), it binds the exposed C-terminal tail of newly folded RbcL in a central cleft, stabilizes that
segment, and bridges RbcL dimers so they assemble into the L8 core. Subsequent RuBisCO small subunit
binding displaces RbcX to yield the L8S8 holoenzyme. When co-expressed in *E. coli*,
RbcX increases accumulation of RbcL/S and improves holoenzyme activity; loss-of-function variants reduce RuBisCO
yield and activity (Onizuka et al., 2004).

In our system, recombinant `psp_rbcX` from *Picosynechococcus* sp. is co-expressed with
`rhc_cbbL` and `rhc_cbbS` to boost L8 core assembly and
L8S8 reconstitution in heterologous *E. coli*. We selected this ortholog because it is
mechanistically well characterized and natively assembles Form I RuBisCO, making it suitable to assemble our
*R. capsulatus* CbbL/CbbS subunits.

##### BioBrick

The `psp_rbcX`–`rhc_cbbS` coding sequences were synthesized as a BioBrick (standard prefix/suffix; IDT)
and subcloned as the middle cistron of a Calvin-module operon. It is co-expressed with `rhc_cbbL` in a bicistronic
unit positioned downstream of `rhc_cbbL`. Transcription of the operon is driven by a single upstream T7–lac regulatory
region (T7 promoter with a lac operator for IPTG-tunable, T7 RNAP–dependent expression). A dedicated RBS (BBa\_B0034) precedes
each CDS, and transcription terminates once after `rhc_cbbS` using the iGEM double terminator BBa\_B0015 (*rrnB* T1 + T7Te).

![psp_rbcX–rhc_cbbS schematic](https://static.igem.wiki/teams/5571/images/danielwet/2.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using the same primer pair:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![psp_rbcX–rhc_cbbS PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/3.webp)

Successful PCR of **psp\_rbcX – rhc\_cbbS BioBrick** with general F/R primers, showing the expected ~1.1 kb band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `rhc_cbbS` insert was cloned into the iGEM backbone pSB1C3. The PCR product (`rhc_cbbS`) and pSB1C3
were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone
was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate
pSB1C3::rhc\_cbbS, transformed into *E. coli* DH5α, selected on chloramphenicol plates, and verified by colony PCR and
EcoRI/PstI diagnostic digest. Final candidate clones were submitted to Plasmidsaurus for sequencing.

#### Assembly of a RuBisCO / RuBisCO-chaperone expression plasmid

Subcloning of `rhc_cbbL–1C3` and `psp_rbcX–rhc_cbbS–1C3` was performed using the following steps.
`rhc_cbbL–1C3` (destination backbone) was digested with AccI/XmaI (Thermo) and PstI FastDigest (Thermo), then
PCR-purified and dephosphorylated with Quick CIP (NEB) to limit self-ligation. In parallel,
`psp_rbcX–rhc_cbbS–1C3` (insert donor) was digested with NarI (NEB) and PstI FastDigest (Thermo) and PCR-purified.
Insert and vector were ligated at a 5:1 (insert:vector) molar ratio to generate pSB1C3::rhc\_cbbL–psp\_rbcX–rhc\_cbbS. Ligation mixes
were transformed into *E. coli* DH5α, plated on chloramphenicol, and screened by colony PCR and EcoRI/PstI diagnostic digest.

Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.

#### csp\_prkA — *Cereibacter sphaeroides* Phosphoribulokinase 1 (UniProt: P12033 · KPPR1\_CERSP)

The *Cereibacter sphaeroides* phosphoribulokinase (`prkA`) is a soluble, cytosolic bacterial Form I-type PRK
that catalyzes the ATP-dependent phosphorylation of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate (RuBP), the committed
substrate for RuBisCO in the CBB cycle: (Ru5P + ATP → RuBP + ADP + H+). By regenerating RuBP from pentose-phosphate
pathway intermediates, `prkA` completes the CBB cycle and sets the RuBP supply rate that gates RuBisCO carboxylation
(Kung et al., 1999).

In our system, `csp_prkA` is co-expressed with `rhc_cbbL`/`rhc_cbbS` (Form I RuBisCO) and β-carbonic
anhydrase to reconstitute the CBB module in *E. coli*. We selected the *C. sphaeroides* ortholog because it is a bacterial
PRK that does not require plant-style thioredoxin redox regulation, simplifying control in *E. coli*, and it has a track record for
robust heterologous expression useful for CBB reconstitution (Kung et al., 1999; Hallenbeck & Kaplan, 1987).

##### BioBrick

The `csp_prkA` coding sequence (UniProt Q3J4A4, KPRK\_RHOSH; *Rhodobacter sphaeroides* phosphoribulokinase) was assembled
as a BioBrick and ordered from IDT. The `csp_prkA` BioBrick is flanked by the standard BioBrick prefix/suffix and includes an RBS
(BBa\_B0034) immediately upstream of the coding sequence. It is intended to be expressed from a T7–lac regulatory cassette as the first gene
in a bicistronic operon, followed by a single downstream gene (e.g., `dci_bCA`).

![csp_prkA schematic](https://static.igem.wiki/teams/5571/images/danielwet/4.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![csp_prkA PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/prka-pcr.avif)

Successful PCR of **csp\_prkA** with general F/R primers, showing the expected ~1.1 kb band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `csp_prkA` insert was cloned into the iGEM backbone pSB1C3. The PCR product (`csp_prkA`) and pSB1C3 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate pSB1C3::csp\_prkA, transformed into *E. coli* DH5α, selected on chloramphenicol plates, and verified by colony PCR and EcoRI/PstI diagnostic digest.

Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.

#### dci\_bCA — *Dolichospermum circinale* β-Carbonic anhydrase (UniProt: A0A0C1N7A1 · A0A0C1N7A1\_9CYAN)

The *Dolichospermum circinale* β-carbonic anhydrase (β-CA) is a zinc metalloenzyme that catalyzes the rapid, reversible hydration of carbon dioxide to bicarbonate:
(CO2 + H2O ⇌ HCO3− + H+). β-CAs are typically small, soluble cytosolic enzymes that assemble as dimers/tetramers and use a Zn2+-bound hydroxide to attack CO2 (Ferraroni, 2024). By accelerating the CO2–bicarbonate interconversion, β-CA maintains near-instantaneous inorganic carbon availability for pathways that specifically use either CO2 (e.g., RuBisCO carboxylation) or HCO3− (e.g., bicarbonate-responsive sensors).

In our work, we chose to recombinantly express β-CA from *Dolichospermum circinale*, a cyanobacterium; cyanobacterial β-CAs are present in carboxysomes, catalyzing the dehydration of HCO3− to CO2 as surrounding CO2 is consumed by RuBisCO (Langella et al., 2022). Additionally, its smaller size reduces stress on the cell without sacrificing its conserved β-CA active site (NCBI, 2025). By co-expressing `dci_bCA` with `rhc_cbbL`/`rhc_cbbS` and `prkA`, our system can mimic efficient autotrophic capabilities through a small number of recombinant genes and without a carboxysome.

##### BioBrick

The `dci_bCA` coding sequence (UniProt Q3M6X9, CAH\_DOLCI; *Dolichospermum circinale* β-carbonic anhydrase) was assembled as a BioBrick and ordered from IDT. The BioBrick is flanked by the standard iGEM prefix/suffix and includes an RBS (BBa\_B0034) immediately upstream of the coding sequence. Transcription terminates with BBa\_B0015 (rrnB T1 + T7Te). The construct is designed for downstream cloning into an IPTG-inducible T7–lacO cassette for co-expression with `csp_prkA` in the second bicistronic operon within the pCBB⁺ megaplasmid.

![dci_bCA schematic](https://static.igem.wiki/teams/5571/images/danielwet/6.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![dci_bCA PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/7.webp)

Successful PCR of **dci\_bCA** with general F/R primers, showing the expected ~955 bp band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `dci_bCA` insert was cloned into the iGEM backbone pSB1C3. The PCR product (`dci_bCA`) and pSB1C3 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate pSB1C3::dci\_bCA, transformed into *E. coli* DH5α, selected on chloramphenicol plates, and verified by colony PCR and EcoRI/PstI diagnostic digest.

![dci_bCA cloning gel](https://static.igem.wiki/teams/5571/images/danielwet/8.webp)

#### Assembly of *prkA* – *bCA* Expression Plasmid

The following workflow was planned to assemble pSB1C3::csp\_prkA–dci\_bCA. The destination backbone (csp\_prkA–1C3) would be digested with AccI/XmaI (Thermo) and PstI FastDigest (Thermo), PCR-purified, and dephosphorylated with Quick CIP (NEB) to limit self-ligation. In parallel, the insert donor (dci\_bCA–1C3) would be digested with NarI (NEB) and PstI FastDigest (Thermo) and PCR-purified. Insert and vector were to be ligated at a 5:1 (insert:vector) molar ratio to generate pSB1C3::csp\_prkA–dci\_bCA. The ligation product would then be transformed into *E. coli* DH5α, selected on chloramphenicol, and screened by colony PCR and EcoRI/PstI diagnostic digest. Any potential clones would be verified by Plasmidsaurus sequencing.

#### Assembly of Final pCBB⁺ Megaplasmid

The subcloning workflow for pSB1C3::rhc\_cbbL–psp\_rbcX–rhc\_cbbS and pSB1C3::csp\_prkA–dci\_bCA was planned as follows. The destination backbone (pSB1C3::rhc\_cbbL–psp\_rbcX–rhc\_cbbS) would be digested with AseI (NEB) and PstI FastDigest (Thermo), PCR-purified, and dephosphorylated with Quick CIP (NEB) to limit self-ligation. In parallel, the insert donor (pSB1C3::csp\_prkA–dci\_bCA) would be digested with NdeI FastDigest (Thermo) and PstI FastDigest (Thermo) to excise the `csp_prkA–dci_bCA` insert, and PCR-purified. Insert and vector were to be ligated at a 3:1 (insert:vector) molar ratio to generate the pCBB⁺ construct. The ligation product would then be transformed into *E. coli* DH5α, selected on chloramphenicol, and screened by colony PCR and EcoRI/PstI diagnostic digest. Any potential clones would be verified by Plasmidsaurus sequencing.

#### psp\_FDH — *Pseudomonas* sp. NAD+-dependent Formate Dehydrogenase (UniProt: P33160 · FDH\_PSESR)

The *Pseudomonas* sp. NAD+-dependent formate dehydrogenase (FDH) catalyzes oxidation of formate to CO2 with reduction of NAD+ to NADH:
(HCOO− + NAD+ → CO2 + NADH + H+). This enzyme is a soluble, cytosolic homodimer (~400 aa per subunit) with an N-terminal Rossmann fold for nucleotide binding; catalysis proceeds by direct hydride transfer from formate to NAD+ at the active site (Tishkov et al., 1993).

In our work, `psp_FDH` is used as a cofactor-recycling module to generate NADH *in situ* from inexpensive formate, whose benign by-product is CO2. Coupling FDH to NADH-dependent steps (e.g., dehydrogenases/reductases) sustains high reducing-equivalent supply without excessive glucose drain. The *Pseudomonas* ortholog was chosen due to its established history of successful recombinant expression in *E. coli*, multiple high-quality crystal structures, and comprehensive biochemical characterization (Tishkov et al., 1993; UniProt P33160).

##### BioBrick

The `psp_FDH` coding sequence (UniProt P33160, FDH\_PSESR; *Pseudomonas* sp. 101 formate dehydrogenase) was assembled as a BioBrick and ordered from IDT. The part is flanked by the standard iGEM prefix/suffix and includes an RBS (BBa\_B0034) immediately upstream of the coding region. Transcription terminates with BBa\_B0015 (rrnB T1 + T7Te). The construct is placed under lac promoter–operator control and is intended for cloning into pSB3T5 as the second plasmid in our two-part autotrophy system to supply FDH expression (enabling NADH-coupled CO2 reduction).

![psp_FDH schematic](https://static.igem.wiki/teams/5571/images/danielwet/9.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![psp_FDH PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/10.webp)

Successful PCR of **psp\_FDH** with general F/R primers, showing the expected ~1.5 kb band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `psp_FDH` insert was cloned into the iGEM backbone pSB3T5. The PCR product (`psp_FDH`) and pSB3T5 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate pSB3T5::psp\_FDH, transformed into *E. coli* DH5α, selected on tetracycline, and verified by colony PCR and EcoRI/PstI diagnostic digest.

### Acetate Production Module

#### Construction of our Acetate Production Module

Our acetate module expresses `ckl_pta` (*Clostridium kluyveri* phosphate acetyltransferase) and `bsu_ackA` (*Bacillus subtilis* acetate kinase) as a recombinant pta–ackA operon for acetate metabolism (acetyl-CoA ⇄ acetyl-phosphate ⇄ acetate).

The operon is assembled on the pRHA-113 backbone for rhamnose-inducible expression. Each coding sequence is preceded by an RBS (BBa\_B0034), and transcription terminates with BBa\_B0015 (rrnB T1 + T7Te).

#### ckl\_pta — *Clostridium kluyveri* Phosphate acetyltransferase (UniProt: A5N801 · A5N801\_CLOK5)

Phosphate acetyltransferase (pta) from *C. kluyveri* catalyzes the reversible conversion of acetyl-CoA and inorganic phosphate to CoA-SH and acetyl phosphate (acetyl-CoA + Pi ⇌ CoA-SH + acetyl phosphate) (Ferry, 2011; UniProt A5N801).

Together with acetate kinase (ackA), pta forms the pta–ackA pathway, channeling excess acetyl-CoA into acetate via acetyl-phosphate. This pathway underlies acetate overflow; when acetyl-CoA accumulates, pta and ackA divert flux toward acetate, preventing toxic buildup and maintaining metabolic balance (Ferry, 2011).

In engineered systems, pta can be co-expressed with ackA to promote acetate secretion or enable efficient co-consumption of acetate (Enjalbert et al., 2017). Activity must be tuned, as excessive acetate can inhibit growth (Pinhal et al., 2019).

We express *C. kluyveri* pta to pair with heterologous *B. subtilis* ackA. In its native physiology, *C. kluyveri* favors the forward pta–ackA reaction during ethanol + acetate chain elongation (Seedorf et al., 2008). Pairing `ckl_pta` with `bsu_ackA` improves kinetic matching, pulling carbon from acetyl-CoA → acetyl-P → acetate, increasing CoA-SH recycling, ATP generation, and acetate export.

##### BioBrick

The `ckl_pta` coding sequence (UniProt A5N801, A5N801\_CLOK5) was assembled as a BioBrick and ordered from IDT. It includes an RBS (BBa\_B0034) and is designed downstream of the rha promoter on pRHA-113, positioned upstream of `bsu_ackA` in a bicistronic operon terminated by BBa\_B0015.

![ckl_pta BioBrick schematic](https://static.igem.wiki/teams/5571/images/danielwet/11.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![ckl_pta PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/12.webp)

##### Cloning

The `ckl_pta` insert was cloned into pRHA-113. The `ckl_pta` PCR product and pRHA-113 were double-digested with XbaI (NEB) and Acc65I (Thermo) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB). Insert and vector were ligated at a 5:1 (insert:vector) ratio to generate pRHA-113::ckl\_pta, transformed into *E. coli* DH5α, selected on ampicillin, and verified by colony PCR and XbaI/Acc65I diagnostic digest.

Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.

#### bsu\_ackA — *Bacillus subtilis* Acetate kinase (UniProt: P37877 · ACKA\_BACSU)

Acetate kinase (AckA) catalyzes acetyl-phosphate + ADP ⇌ acetate + ATP and is a homodimeric ASKHA-family enzyme with active sites at the dimer interface (Buss et al., 2001; Ingram-Smith et al., 2015).

With pta, AckA completes the pta–ackA node to direct excess acetyl-CoA to acetate (Enjalbert et al., 2017). Acetyl-phosphate can also act as a signaling metabolite in global regulation (Klein et al., 2007). Tuning is required to avoid growth inhibition from acetate (Pinhal et al., 2019).

We express *B. subtilis* AckA (rather than *E. coli* AckA) to bias flux toward the forward reaction (acetyl-P + ADP → acetate + ATP) and exploit interspecies kinetic diversity that can better sustain net acetate production under our conditions.

##### BioBrick

The `bsu_ackA` coding sequence (UniProt P37877, ACKA\_BACSU) was assembled as a BioBrick with an RBS (BBa\_B0034). It is positioned downstream of `ckl_pta` on pRHA-113 under the rhaBAD promoter, with BBa\_B0015 terminating transcription.

![bsu_ackA BioBrick schematic](https://static.igem.wiki/teams/5571/images/danielwet/13.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![bsu_ackA PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/14.webp)

Successful PCR of **bsu\_ackA** with general F/R primers, showing the expected ~1.5 kb band in all lanes (0.8% TAE agarose gel; ladder at left).

##### Cloning

The `bsu_ackA` insert was cloned into the pRHA-113 backbone. The `bsu_ackA` PCR product and pRHA-113 were double-digested with XbaI (NEB) and Acc65I (Thermo) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated at a 5:1 (insert:vector) molar ratio to generate pRHA-113::bsu\_ackA, transformed into *E. coli* DH5α, selected on ampicillin plates, and verified by colony PCR and XbaI/Acc65I diagnostic digest.

Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.

### Assembly of Final pRHA–pta–ackA Plasmid

The subcloning workflow for pRHA113::ckl\_pta and pRHA113::bsu\_ackA was planned as follows. The destination backbone (pRHA113::ckl\_pta) would be digested with AccI/XmaI (Thermo) and Acc65I (Thermo), PCR-purified, and dephosphorylated with Quick CIP (NEB) to limit self-ligation. In parallel, the insert donor (pRHA113::bsu\_ackA) would be digested with NarI (NEB) and Acc65I (Thermo) to excise the `bsu_ackA` insert, and PCR-purified. Insert and vector were to be ligated at a 5:1 (insert:vector) molar ratio to generate the pRHA–pta–ackA construct. The ligation product would then be transformed into *E. coli* DH5α, selected on ampicillin, and screened by colony PCR and XbaI/Acc65I diagnostic digest. Any potential clones would be verified by Plasmidsaurus sequencing.

### Final Transformation of BL21(DE3) Autotrophic Acetate Producer (Strain 1)

*The following series of transformations and tests was planned in the event that successful clones were obtained.*

BL21(DE3) competent cells would be transformed in stages:

1. **Stage 1 — pCBB⁺ (CmR)**: Transform with pCBB⁺ and select on chloramphenicol plates. Screen colonies by diagnostic digest and/or colony PCR. Only verified clones proceed.
2. **Stage 2 — pSB3T5–FDH (TetR)**: Transform the verified pCBB⁺ strain with pSB3T5–FDH. Select on Cm + Tet plates and re-verify by diagnostic digest/colony PCR.
3. **Stage 3 — pRHA–pta–ackA (AmpR)**: Transform the double-plasmid strain with pRHA–pta–ackA. Select on Cm + Tet + Amp plates and confirm the triple-plasmid strain by diagnostic digest and/or colony PCR.

## Strain 2: PHBV Producer

### Core Design

#### Goals

* Produce PHBV in *E. coli* from acetate via PHB core genes + endogenous propionyl-CoA (tdcB/tdcE) for PHV incorporation.
* Boost yield with CRISPRi knockdown of *gltA* (citrate synthase) using SadCas9.

PHBV module (pPHBV + pCo⁺ + CRISPRi)

The PHBV Producer Module is the downstream half of our two-strain system: it consumes acetate secreted by Strain 1 and converts it into PHBV. Rather than using a native PHB producer (e.g., *Cupriavidus necator* or *I. basilensis*), we propose engineering *E. coli* to produce PHBV by (i) introducing the essential PHB biosynthetic genes, (ii) enabling endogenous propionyl-CoA formation to permit PHV incorporation (via the threonine → 2-ketobutyrate → propionyl-CoA route mediated by *tdcB/tdcE*), and (iii) applying CRISPRi to knock down competing flux (e.g., *gltA*/citrate synthase) to improve yield.

#### Construction of our PHBV Production Module

Our PHBV module expresses **bktB** (β-ketothiolase, *Haloferax mediterranei*), **phaB** (acetoacetyl-CoA reductase, *Synechocystis sp.*), and **phaC** (PHA synthase, *Allochromatium vinosum*) for PHB biosynthesis; **tdcB/tdcE** (L-threonine dehydratase from *Shigella flexneri* and a glycyl-radical pyruvate formate-lyase family enzyme from *E. coli*) to enable endogenous propionyl-CoA formation and thereby permit PHV incorporation; and the thioesterases **yciA** (*S. flexneri*) and **tesB** (*Y. pestis*) to maintain free 3HB/3HV pools and prevent CoA sequestration. Furthermore, we use SadCas9 with an sgRNA targeting the transcription-start-site (TSS)–proximal region of *E. coli* *gltA* to repress transcription via CRISPRi.

These genes are expressed using a three-plasmid system: (i) **pPHBV**, which carries the PHB/PHBV biosynthesis operon; (ii) the companion plasmid **pCo⁺**, which enables endogenous propionyl-CoA formation through the threonine → 2-ketobutyrate → propionyl-CoA route (*tdcB*, *tdcE*) and incorporates *tesB* and *yciA* to support 3HB/3HV availability and CoA balance; and (iii) **pSB1A3-sgRNA–SadCas9**, a CRISPRi module targeting endogenous *E. coli* *gltA* (citrate synthase) to reduce acetyl-CoA flux into the citric acid cycle and divert acetyl-CoA toward polymer production.

1. **pPHBV (TetR, p15A ori)** was assembled in pSB3T5 (BioBrick prefix/suffix) by cloning a composite, T7–lacO–driven operon. A single polycistronic cassette—`ssp_phaB–avi_phaC–hme_bktBα–hme_bktBβ`—is driven by a T7–lacO regulatory region, includes an RBS (BBa\_B0034) upstream of every coding sequence (CDS), and terminates with the iGEM double terminator BBa\_B0015 (*rrnB* T1 + T7Te).
2. **pCo⁺ (CmR, pSC101 ori)** was cloned in pSB4C5 and supports propionyl-CoA biosynthesis and CoA balance. The construct contains `tdcB–tdcE` (L-threonine dehydratase and glycyl-radical pyruvate formate-lyase family enzyme), `saz_aCA` (α-carbonic anhydrase, *S. azorense*), `hsa_thsaccat` (soluble adenylyl cyclase, human *ADCY10*), and the thioesterases `tesB` (*Y. pestis*) and `yciA` (*S. flexneri*). Expression is governed by a lac promoter–operator system for IPTG-inducible control, with each CDS preceded by an RBS (BBa\_B0034) and terminated by BBa\_B0015.
3. **pPHBV vs. pPHBV⁺ (CRP box)**: The pPHBV plasmid exists in two versions that differ only by a CRP box placed immediately upstream of the T7–lacO element driving the first gene, *ssp\_phaB*. In pPHBV, transcription is governed solely by IPTG-inducible T7–lacO control. In the CRP-box variant (pPHBV⁺), the upstream CRP-binding site enables additional regulation through intracellular cAMP–CRP. When co-expressed with pCo⁺, α-carbonic anhydrase elevates intracellular bicarbonate (HCO3−), stimulating *ADCY10* to generate cAMP, which activates CRP and enhances transcription of the *phaB–phaC–bktBα/β* operon in response to CO2/HCO3− levels.
4. **CRPbox\_msGFP2 reporter**: To assay transcriptional increase from the CRP box, we designed a *CRPbox\_msGFP2* fluorescent reporter (see project description).

### Individual Part Characterization & Data

#### ssp\_phaB — *Synechocystis sp.* acetoacetyl-CoA reductase (UniProt: P73826 · PHAB\_SYNY3)

The *Synechocystis sp.* acetoacetyl-CoA reductase (phaB) is an SDR-family dehydrogenase that catalyzes the stereospecific, NADPH-dependent reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, supplying the PHB monomer downstream of phaA/bktB in the canonical phaA/phaB/phaC pathway (Liu et al., 2015).

In heterologous hosts, recombinant expression of *Synechocystis* phaB confers robust PHB production (up to ~12% CDW depending on carbon source), making it a reliable monomer producer. We leverage this precedent to drive monomer formation in our PHB/PHBV module.

##### BioBrick

The `ssp_phaB` coding sequence (UniProt P73826) was assembled as a BioBrick and ordered from IDT. The part is flanked by the standard iGEM prefix/suffix and includes an RBS (BBa\_B0034). Transcription is driven by a T7–lacO cassette and terminates with T7Te. This is the first gene in the pPHBV operon.

![ssp_phaB construct map](https://static.igem.wiki/teams/5571/images/danielwet/15.webp)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![ssp_phaB PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/wetlabphab.webp)

Successful PCR of **ssp\_phaB** with general F/R primers, ~900 bp (0.8% TAE agarose; ladder at left).

##### Cloning

The `ssp_phaB` insert was cloned into the iGEM backbone pSB3T5. The PCR product and pSB3T5 were double-digested with EcoRI FD and PstI FD, purified, and the backbone dephosphorylated with Quick CIP. A 3:1 ligation generated pSB3T5::`ssp_phaB`, transformed into *E. coli* DH5α, selected on tetracycline, and verified by colony PCR and EcoRI/PstI digest. Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.

#### CRPbox\_T7-phaB (*Synechocystis sp.*)

This composite expresses *Synechocystis sp.* *phaB*, an SDR enzyme that uses NADPH to reduce acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, supplying the 3HB monomer for PHB/PHBV biosynthesis.

A CRP (cAMP receptor protein) binding site (“CRP box”) is inserted immediately upstream of the promoter driving *phaB*. When intracellular cAMP rises, CRP–cAMP binds and activates transcription, coupling *phaB* expression to cAMP signaling. In our system, this links *phaB* output to CO2/HCO3− status via the sAC module (higher bicarbonate → more cAMP → stronger *phaB* expression), allowing carbon-responsive tuning of 3HB supply.

##### BioBrick

The `CRPbox_T7-phaB` variant places a CRP binding site immediately upstream of the T7–lacO regulatory cassette, enabling cAMP-CRP–responsive modulation of the promoter. Flanked by the standard iGEM prefix/suffix with an RBS (BBa\_B0034) directly upstream of the CDS.

![CRPbox_T7-phaB construct map](https://static.igem.wiki/teams/5571/images/danielwet/16.webp)

#### CRPbox\_T7-phaB — PCR & Cloning

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![CRPbox_T7-phaB PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/17.webp)

Successful PCR of **CRPbox\_T7-phaB**, ~930 bp (0.8% TAE agarose; ladder at left).

##### Cloning

The `CRPbox_T7-phaB` insert was cloned into pSB3T5. The PCR product and pSB3T5 were double-digested (EcoRI FD, PstI FD), purified, and the backbone dephosphorylated with Quick CIP. A 3:1 ligation generated pSB3T5::`CRPbox_T7-phaB`, transformed into *E. coli* DH5α, selected on tetracycline, and verified by colony PCR and EcoRI/PstI digest; candidates were submitted for Plasmidsaurus sequencing.

#### avi\_phaC — *Allochromatium vinosum* PHA synthase (UniProt: P45370)

The *A. vinosum* PhaC is the catalytic subunit of a Class III PHA synthase (PhaC–PhaE heterodimer) that polymerizes (R)-3-hydroxyacyl-CoA thioesters into PHA, releasing CoA (Lawrence et al., 2005). Catalysis uses an active-site Cys with His/Asp support and proceeds via a covalent acyl–enzyme intermediate; the enzyme is enantioselective for (R) monomers (Zher Neoh et al., 2022). Substrate scope supports PHB and PHBV formation with suitable 3HB/3HV supply.

##### BioBrick

The `avi_phaC` CDS (UniProt P45370) was synthesized as a BioBrick (std. prefix/suffix) with RBS (BBa\_B0034). In pPHBV, `avi_phaC` is the second cistron (downstream of `ssp_phaB`, upstream of `hme_bktB`).

![avi_phaC construct map](https://static.igem.wiki/teams/5571/images/danielwet/screenshot-2025-10-07-231513.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![avi_phaC PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/19.webp)

Successful PCR of **avi\_phaC**, ~1.2 kb (note: smear in lane 3 due to undissolved agarose).

##### Cloning

`avi_phaC` was cloned into pSB3T5 via EcoRI/PstI, backbone dephosphorylated, 3:1 ligation, DH5α transformation, Tet selection, and EcoRI/PstI verification; candidates submitted for sequencing.

#### hme\_bktB — *Haloferax mediterranei* β-ketothiolase (α/β)

*H. mediterranei* uses heterotetrameric thiolases (α/β) to initiate PHB/V biosynthesis. The system condenses acetyl-CoA→acetoacetyl-CoA and also accepts propionyl-CoA to produce 3-ketovaleryl-CoA, enabling 3HV incorporation. Compared with canonical bacterial PhaA, `bktB` supports broader substrate scope and HV diversification.

##### BioBrick

The `hme_bktB` CDS (α/β) was assembled as a BioBrick (std. prefix/suffix) with RBS (BBa\_B0034). In the pPHBV operon it is the third cistron; transcription terminates with BBa\_B0015.

![hme_bktB construct map](https://static.igem.wiki/teams/5571/images/danielwet/20.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![hme_bktB PCR gel (placeholder)](https://static.igem.wiki/teams/5571/images/danielwet/bktb-pcr.avif)

Successful PCR of **hme\_bktB**, ~1.8 kb (0.8% TAE agarose; ladder at left).

##### Cloning

`hme_bktB` was cloned into pSB3T5 (EcoRI/PstI), backbone dephosphorylated, 3:1 ligation, DH5α transformation, Tet selection, EcoRI/PstI verification; candidates submitted for sequencing.

![hme_bktB cloning digest](https://static.igem.wiki/teams/5571/images/danielwet/21.webp)

#### Assembly of Final pPHBV / pPHBV⁺

1. **Step 1:** Linearize pSB3T5::`ssp_phaB` with AccI/XmiI and PstI; purify & dephosphorylate. Excise `avi_phaC` from pSB3T5::`avi_phaC` with NarI/PstI; purify. Ligate (3:1) → pSB3T5::`T7–lacO–ssp_phaB–avi_phaC`; transform DH5α; Tet select; EcoRI/PstI screen; sequence.
2. **Step 2:** Linearize new destination with AseI/VspI and PstI; purify & dephosphorylate. Excise `hme_bktB` from pSB3T5::`hme_bktB` with NdeI/PstI; purify. Ligate (3:1) → pPHBV; transform DH5α; Tet select; EcoRI/PstI screen; sequence.
3. **pPHBV⁺ variant:** identical workflow but starting from pSB3T5::`CRPbox_T7-phaB`.

#### sfl\_tdcB — *Shigella flexneri* L-threonine dehydratase (catabolic)

`tdcB` converts L-threonine → 2-ketobutyrate + NH4+ (anaerobic), feeding the HV branch (with TdcE + BktB) toward 3-ketovaleryl-CoA.

##### BioBrick

The `sfl_tdcB` CDS (UniProt P0AGF9) was assembled as a BioBrick with RBS (BBa\_B0034); first cistron of the propionyl-CoA cassette on pSB4C5 (lac/ IPTG-inducible), terminating with BBa\_B0015.

![sfl_tdcB construct map](https://static.igem.wiki/teams/5571/images/danielwet/22.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![sfl_tdcB PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/23.webp)

Successful PCR of **sfl\_tdcB**, ~1.3 kb.

##### Cloning

`sfl_tdcB` was cloned into pSB4C5 via EcoRI/PstI, backbone dephosphorylated, 3:1 ligation → pSB4C5::`sfl_tdcB`; DH5α transform; Cm select; EcoRI/PstI verify.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### eco\_tdcE — *E. coli* pyruvate formate-lyase family (propionyl-CoA)

`tdcE` cleaves 2-ketobutyrate → propionyl-CoA + formate (and pyruvate → acetyl-CoA + formate) under anaerobic conditions, supplying the C3 precursor for 3HV incorporation.

##### BioBrick

The `eco_tdcE` CDS (UniProt P42632) was assembled as a BioBrick with RBS (BBa\_B0034); second cistron of the `tdcB–tdcE` operon on pSB4C5 (lac/ IPTG-inducible), with BBa\_B0015 terminator.

![eco_tdcE construct map](https://static.igem.wiki/teams/5571/images/danielwet/24.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![eco_tdcE PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/26.webp)

Successful PCR of **eco\_tdcE**, ~2.6 kb.

##### Cloning

`eco_tdcE` was cloned into pSB4C5 (EcoRI/PstI), backbone dephosphorylated, 3:1 ligation → pSB4C5::`eco_tdcE`; DH5α transform; Cm select; EcoRI/PstI verify.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### saz\_aCA — *Sulfurihydrogenibium azorense* α-carbonic anhydrase

`saz_aCA` rapidly hydrates CO2 ⇄ HCO3−, elevating intracellular bicarbonate to activate soluble adenylyl cyclase (ADCY10), linking inorganic carbon to cAMP signaling and CRP-responsive promoters.

##### BioBrick

The `saz_aCA` CDS (UniProt C1DTU5) was assembled as a BioBrick (RBS BBa\_B0034; std. prefix/suffix) with BBa\_B0015 terminator.

![saz_aCA construct map](https://static.igem.wiki/teams/5571/images/danielwet/27.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![saz_aCA PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/28.webp)

Successful PCR of **saz\_aCA**, ~1.0 kb.

##### Cloning

`saz_aCA` → pSB4C5 (EcoRI/PstI), 3:1 ligation, DH5α transform, Cm select, EcoRI/PstI verify.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### hsa\_thsaccat — human soluble adenylyl cyclase (ADCY10)

Human sAC (ADCY10) produces cAMP from ATP in response to HCO3−/Ca2+, independent of GPCRs, making it a direct CO2/HCO3− sensor for coupling to CRP-responsive control.

##### BioBrick

The `hsa_thsaccat` CDS (UniProt Q96PN6) was assembled as a BioBrick (RBS BBa\_B0034; std. prefix/suffix). Intended for cloning into an inducible cassette (e.g., lac or T7–lacO); terminator provided by destination cassette. Paired with `saz_aCA` to generate a bicarbonate-activated sAC → cAMP signal.

![ADCY10 construct map](https://static.igem.wiki/teams/5571/images/danielwet/29.webp)

#### hsa\_thsACcat — human soluble adenylyl cyclase (ADCY10)

##### PCR

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![hsa_thsACcat PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/30.webp)

Successful PCR of **hsa\_thsACcat**, ~1.7 kb (0.8% TAE agarose; ladder at left).

##### Cloning

The `hsa_thsACcat` insert was cloned into pSB4C5. PCR product and pSB4C5 were double-digested (EcoRI FD, PstI FD), purified; backbone dephosphorylated with Quick CIP. A 3:1 ligation yielded pSB4C5::`hsa_thsACcat`, transformed into *E. coli* DH5α, Cm-selected, and verified by colony PCR and EcoRI/PstI digest.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### ype\_tesB — *Yersinia pestis* acyl-CoA thioesterase II

Acyl-CoA thioesterase II (TesB) hydrolyzes acyl-CoA → free acid + CoA-SH, aiding CoA recycling and relieving inhibitory acyl-CoA buildup. Overexpressed TesB can raise 3HB titers and support PHB production when balanced with PhaA/PhaB (Ku & Lan, 2018).

##### BioBrick

The `ype_tesB` CDS (UniProt A0A3N4BCC7) was assembled as a BioBrick with RBS (BBa\_B0034) under a lac–lacO cassette; terminates with BBa\_B0015.

![ype_tesB construct map](https://static.igem.wiki/teams/5571/images/danielwet/31.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![ype_tesB PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/32.webp)

Successful PCR of **ype\_tesB**, ~1.7 kb.

##### Cloning

`ype_tesB` → pSB4C5 via EcoRI/PstI; backbone dephosphorylated; 3:1 ligation → pSB4C5::`ype_tesB`; DH5α transform; Cm select; EcoRI/PstI verify.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### sfl\_yciA — *Shigella flexneri* acyl-CoA thioesterase

YciA (hot-dog fold thioesterase) broadly hydrolyzes acyl-CoAs including acetyl-CoA, acetoacetyl-CoA, and (R)/(S)-3HB-CoA, buffering CoA homeostasis and stabilizing upstream flux.

##### BioBrick

The `sfl_yciA` CDS was assembled as a BioBrick with RBS (BBa\_B0034) under a lac–lacO cassette; BBa\_B0015 terminator.

![sfl_yciA construct map](https://static.igem.wiki/teams/5571/images/danielwet/33.webp)

##### PCR

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![sfl_yciA PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/34.webp)

Successful PCR of **sfl\_yciA**, ~700 bp.

##### Cloning

`sfl_yciA` → pSB4C5 via EcoRI/PstI; backbone dephosphorylated; 3:1 ligation → pSB4C5::`sfl_yciA`; DH5α transform; Cm select; EcoRI/PstI verify.

*Despite multiple iterations, no confirmed clones were recovered within the experimental timeframe.*

#### Assembly of final pCo⁺ plasmid

Each subcloning event is performed separately, then combined:

1. **tdcB–tdcE operon:** Linearize pSB4C5::`eco_tdcE` with NarI + PstI; purify & dephosphorylate. Excise `sfl_tdcB` from pSB4C5::`sfl_tdcB` with AccI/XmiI + PstI; purify. Ligate (3:1) → pSB4C5::`sfl_tdcB–eco_tdcE`; transform DH5α; Cm select; EcoRI/PstI screen; sequence.
2. **hsa\_thsACcat–saz\_aCA module:** Linearize pSB4C5::`hsa_thsACcat` with AccI/XmiI + PstI; purify & dephosphorylate. Excise `saz_aCA` from pSB4C5::`saz_aCA` with NarI + PstI; purify. Ligate (3:1) → pSB4C5::`hsa_thsACcat–saz_aCA`; transform; Cm select; screen; sequence.
3. **tesB–yciA module:** Linearize pSB4C5::`ype_tesB` with AccI/XmiI + PstI; purify & dephosphorylate. Excise `sfl_yciA` from pSB4C5::`sfl_yciA` with NarI + PstI; purify. Ligate (3:1) → pSB4C5::`ype_tesB–sfl_yciA`; transform; Cm select; screen; sequence.

**Final joins:**

1. Linearize pSB4C5::`sfl_tdcB–eco_tdcE` with AseI/VspI + PstI; purify & dephosphorylate. Excise `hsa_thsACcat–saz_aCA` with NdeI + PstI; ligate (3:1) → pSB4C5::`sfl_tdcB–eco_tdcE–hsa_thsACcat–saz_aCA`; transform; Cm select; screen; sequence.
2. Linearize the above with AseI/VspI + PstI; purify & dephosphorylate. Excise `ype_tesB–sfl_yciA` with NdeI + PstI; ligate (3:1) → **pCo⁺** (pSB4C5::`sfl_tdcB–eco_tdcE–hsa_thsACcat–saz_aCA–ype_tesB–sfl_yciA`); transform DH5α; Cm select; EcoRI/PstI screen; sequence.

### CRISPRi Module — SadCas9 + sgRNA

#### CRISPRi module — SadCas9 + sgRNA (gltA)

pSB1A3 backbone (AmpR, pUC ori). sgRNA driven by J23119; SadCas9 driven by a pTet-derived promoter (BBa\_K3171173; aTc-inducible). Each CDS has RBS (BBa\_B0034) and terminates with BBa\_B0015.

* Reporter: `gltA`-recognition sequence placed TSS-proximal upstream of `msGFP2` on a separate plasmid to assay knockdown vs. controls.

##### SadCas9 background

*S. aureus* dCas9 (SadCas9) retains sequence-programmable binding (PAM 5′-NNGRRT-3′) without nuclease activity. Binding blocks transcription; TSS-proximal, non-template strand targeting yields strongest repression. Its compact size (~3.2 kb) eases delivery vs. SpdCas9 (~4.1 kb).

##### BioBrick

SadCas9 (UniProt A0A386IRG9) was ordered in two parts (SadCas9pt1/pt2), assembled as a BioBrick (std. prefix/suffix, RBS BBa\_B0034) and placed under pTet-derived control; terminates with BBa\_B0015.

![SadCas9 part 1](https://static.igem.wiki/teams/5571/images/danielwet/35.webp)

![SadCas9 assembly schematic](https://static.igem.wiki/teams/5571/images/danielwet/37.webp)

![SadCas9 part 2](https://static.igem.wiki/teams/5571/images/danielwet/38.webp)

#### SadCas9 (continued)

![SadCas9 schematic / part image](https://static.igem.wiki/teams/5571/images/danielwet/38.webp)

##### PCR — SadCas9pt1

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![SadCas9pt1 PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/sadcas9part1-pcr.avif)

Successful PCR of **SadCas9pt1**, ~1.7 kb (0.8% TAE agarose; ladder at left).

##### PCR — SadCas9pt2

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![SadCas9pt2 PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/sadcas9part2-pcr.avif)

Successful PCR of **SadCas9pt2**, ~1.8 kb (0.8% TAE agarose; ladder at left).

##### Cloning — SadCas9pt1

The **SadCas9pt1** insert was cloned into the iGEM backbone pSB1A3. The PCR product (SadCas9pt1) and pSB1A3 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate
*pSB1A3::SadCas9pt1*, transformed into *E. coli* DH5α, selected on ampicillin plates, and verified by colony PCR and EcoRI/PstI diagnostic digest.

*Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.*

##### Cloning — SadCas9pt2

The **SadCas9pt2** insert was cloned into the iGEM backbone pSB1A3. The PCR product (SadCas9pt2) and pSB1A3 were double-digested with EcoRI FD and PstI FD (Thermo Fisher Scientific) and purified using a PCR cleanup kit (NEB). The backbone was dephosphorylated with Quick CIP (NEB) to minimize self-ligation. Insert and vector were ligated in a 3:1 ratio to generate *pSB1A3::SadCas9pt2*, transformed into *E. coli* DH5α, selected on ampicillin plates, and verified by colony PCR and EcoRI/PstI diagnostic digest. Final candidate clones were submitted to Plasmidsaurus for sequencing.

![SadCas9pt2 cloning gel / map](https://static.igem.wiki/teams/5571/images/danielwet/39.webp)

#### Assembly of Final SadCas9 Construct

The destination backbone *pSB1A3::SadCas9pt2* would be linearized with EcoRI FastDigest (Thermo) and BstAPI (NEB), PCR-purified, and dephosphorylated with Quick CIP (NEB) to limit self-ligation. In parallel, the insert donor *pSB1A3::SadCas9pt1* would be digested with EcoRI FastDigest (Thermo) and BstAPI (NEB) to excise the SadCas9pt1 fragment and PCR-purified. Insert and vector would then be ligated at a 3:1 (insert:vector) molar ratio to generate *pSB1A3::SadCas9*. Ligation products would be transformed into *E. coli* DH5α, selected on ampicillin,
and screened by colony PCR and EcoRI/PstI diagnostic digest; candidate clones would be sequence-verified (Plasmidsaurus).

*Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.*

#### sgRNA — gltA (citrate synthase) targeting

The single-guide RNA (sgRNA) fuses crRNA (spacer) and tracrRNA (scaffold) to direct dCas9 to the target locus. For Sa-dCas9, a 5′-NNGRRT-3′ PAM is required. Targeting the promoter/TSS-proximal region enables strong CRISPRi repression.

We express a *Staphylococcus aureus*–specific sgRNA scaffold with ~21-nt spacers adjacent to 5′-NNGRRT-3′ PAM sites near the *gltA* TSS. Knockdown is first quantified via a *gltA* recognition (gltArec)–msGFP2 reporter assay.

##### BioBrick

For assay purposes, sgRNA was fused to *gltArec*–msGFP2; this reporter cassette is removed in the final construct. The *gltArec*–msGFP2 reporter was assembled as a BioBrick (std. prefix/suffix, RBS BBa\_B0034), lac–lacO driven, and terminates with BBa\_B0015.

![gltArec–msGFP2 schematic](https://static.igem.wiki/teams/5571/images/danielwet/40.webp)

##### PCR — sgRNA–gltArec–msGFP2

Standardized 5′/3′ primer sites were added across constructs so they can be PCR-amplified using a single primer pair we designed:

* General Forward — 5′ CGGACTTTCGGTCAACCACAATTC 3′
* General Reverse — 5′ GGCAGCAGTGCATTGAAACTTC 3′

![sgRNA–gltArec–msGFP2 PCR gel](https://static.igem.wiki/teams/5571/images/danielwet/41.webp)

Successful PCR of **sgRNA–gltArec–msGFP2**, ~1.4 kb.

##### Cloning — sgRNA–gltArec–msGFP2

The **sgRNA–gltArec–msGFP2** insert was cloned into pSB1A3. PCR product and pSB1A3 were double-digested with EcoRI FD and PstI FD, purified; backbone dephosphorylated with Quick CIP. A 3:1 ligation yielded *pSB1A3::sgRNA–gltArec–msGFP2*, transformed into *E. coli* DH5α, Amp-selected, and verified by colony PCR and EcoRI/PstI digest.

*Despite multiple iterations, no confirmed clones were recovered within the duration of the experimental timeframe.*

#### Assembly of Final pSB1A3–SadCas9–sgRNA Plasmid

The destination backbone *pSB1A3::SadCas9* would be linearized with NarI (NEB) and EcoRI FastDigest (Thermo), PCR-purified, and dephosphorylated with Quick CIP (NEB). In parallel, the insert donor *pSB1A3::sgRNA–gltArec–msGFP2* would be digested with AccI/XmiI (Thermo) and EcoRI FastDigest (Thermo) to excise the sgRNA fragment and PCR-purified. Insert and vector would then be ligated at a 3:1 (insert:vector) molar ratio to generate *pSB1A3::sgRNA–SadCas9*. Ligation products would be transformed into *E. coli* DH5α, Amp-selected, and screened by colony PCR and EcoRI/PstI digest; candidate clones would be sequence-verified (Plasmidsaurus).

#### Final Transformation of BL21(DE3) PHBV Producer Strain 2

*The following series of transformations and tests was planned in the event that successful clones were obtained.*

1. **Stage 1 — pPHBV⁺ (TetR)**: Transform with pPHBV⁺ and select on
   tetracycline plates. Screen colonies by diagnostic digest and/or colony PCR. Only verified clones proceed.
2. **Stage 2 — pCo⁺ (CmR)**: Transform the verified pPHBV⁺ strain with pCo⁺. Select on Cm + Tet plates and re-verify by diagnostic digest/colony PCR.
3. **Stage 3 — pSB1A3–sgRNA–SadCas9 (AmpR)**: Transform the double-plasmid strain with pSB1A3–sgRNA–SadCas9. Select on Cm + Tet + Amp plates and confirm the triple-plasmid strain by diagnostic digest and/or colony PCR.

### Protein Characterization

#### Protein Quantification: T7-CRP msGFP2 (monomeric superfolder Green Fluorescent Protein 2)

**Protein:** msGFP2

**Expected MW:** 26.2 kDa

**Reference Standards:** recombinant albumin (50 ng/µL, 10 ng/µL)

**Load Volume:** 10 µL per lane

1. Gel Densitometry – Absolute Target Protein Mass

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/protein-gel-1.avif)

**4–20% SDS–PAGE.** Lane 1: uninduced control lysate; Lanes 2–5: lysates induced with **40, 85, 190, 400 μM IPTG (respectively); Lane 6: molecular weight ladder; Lane 8: 50 ng albumin standard; Lane 10: 10 ng albumin standard. (10 μL loaded per lane.)**

2. Bradford Assay–Total Protein Concentration

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/graph-2.avif)

3. Integration of Gel and Bradford Data

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/msgfp2-integration-table.avif)

Expression of **7–lacO–msGFP2** is reliable but low in abundance relative to host proteins. Transcription/translation appear consistent, and lane-to-lane differences are driven mainly by total-protein background. This is somewhat surprising for a T7-driven cassette, which is typically strong, and likely reflects sub-optimal induction/conditions (e.g., IPTG/T7 RNAP level, temperature, growth phase, or protein solubility) rather than failure of the promoter. Nevertheless, we believe protein levels are sufficient to quantify msGFP2 fluorescence in assays comparing conditions with enhanced CRP binding versus no CRP binding.

#### Protein Quantification: T7-lacO rhc\_cbbL (Form I RuBisCO Large Subunit)

**Protein:** rhc\_cbbL

**Expected MW:** 53 kDa

**Reference Standards:** recombinant albumin (50 ng/µL)

**Load Volume:** 10 µL per lane

Gel Densitometry – Relative Target Protein Mass

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/protein-gel-2.avif)

**4–20% SDS–PAGE.** Lane 1: uninduced control lysate; Lanes 2–5: lysates induced with **0, 85, 190, 400 μM IPTG (respectively); Lane 6: molecular weight ladder; Lane 8: 50 ng albumin standard; Lane 10: 10 ng albumin standard. (10 μL loaded per lane.)**

2. Bradford Assay–Total Protein Concentration

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/graph-3.avif)

3. Integration of Gel and Bradford Data

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/cbbl-integration-table.avif)

rhc\_cbbL is detectable but **very low-abundance on SDS–PAGE.** A likely explanation is misfolding/aggregation of the RuBisCO large subunit when expressed without its folding partners (e.g., RbcX and/or GroEL/ES). Misfolded cbbL tends to form insoluble inclusion bodies—potentially toxic—so little soluble protein of the expected ~53 kDa remains despite the T7–lacO expression cassette.

(Implications: co-express RbcX ± CbbS, or add GroEL/ES; lower induction temperature/IPTG, or use solubility-enhancing tags to improve recovery.)

#### Protein Quantification: saz\_αCA–4C5 (α-Carbonic Anhydrase)

**Protein:** saz\_αCA

**Expected MW:** 29.3 kDa

**Reference Standards:** recombinant albumin (50 ng/µL, 10 ng/µL)

**Load Volume:** 10 µL per lane

Gel Densitometry – Absolute Target Protein Mass

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/protein-gel-3.avif)

**4–20% SDS–PAGE.** Lane 1: uninduced control lysate; Lanes 2–5: lysates induced with **0, 85, 190, 400 μM IPTG (respectively); Lane 6: molecular weight ladder; Lane 8: 50 ng albumin standard; Lane 10: 10 ng albumin standard. (10 μL loaded per lane.)**

→ values obtained directly from the calibrated gel using the two albumin standards

2. Bradford Assay–Total Protein Concentration

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/graph-1.avif)

3. Integration of Gel and Bradford Data

![geldense](https://static.igem.wiki/teams/5571/images/danielwet/aca-integration-table.avif)

4. Carbonic Anhydrase Activity

![geldense](https://static.igem.wiki/teams/5571/images/exp-revisions/wilbur-anderson-assay-time-to-2-ph-unit-drop.avif)
![IPTG Induction Conc](https://static.igem.wiki/teams/5571/images/exp-revisions/specific-activity-units-mg-iptg-induction-concentrations.avif)

A Wilbur–Anderson assay was performed to quantify carbonic anhydrase activity in crude lysates from cultures induced with varying IPTG concentrations (40, 85, 190, and 400 μM), alongside an uninduced control. Enzyme activity was measured as the **time required for a 2-unit drop in pH**, with shorter times indicating higher catalytic activity. The boxplot (top) shows a clear decrease in reaction time with increasing IPTG induction, demonstrating that higher induction levels produce more active enzyme.

Specific enzyme activity (units/mg) was calculated using the Wilbur–Anderson formula:

![IPTG Induction Conc](https://static.igem.wiki/teams/5571/images/exp-revisions/units-mg-equation.avif)

where T0 is the uncatalyzed control time, and T is the observed reaction time. Median specific activities increased monotonically with IPTG concentration, rising from **1230 units/mg (40 μM) to 2620 units/mg (400 μM)** (bottom panel). Together, these results show that IPTG induction strongly enhances CA expression and catalytic activity in a dose-dependent manner.

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