iGEM 2025 — Team wellington-shanghai

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![Notebook](https://static.igem.wiki/teams/5682/model/notebook.webp)

# Lab Log

A comprehensive documentation of our experimental journey, research progress, and discoveries throughout the Barnacure project development.

#### Timeline

* [June 2025](#june)
* [August 2025](#august)
* [September 2025](#september)

### June 2025

June 9

Logged by Claire Park

▼

👥 Members Present

**08:45 ~ 12:45:** Jackson, David

**08:45 ~ 15:15:** Claire, Asger, JingJing, Zahara, William, Joanna, Jayden Leung

**14:15 ~ 15:15:** Evelyn, Lifu, Jayden Liu

🔬 Activities

* Mr. Edwards introduced iGEM medal criteria, discussed special prize categories and submission village
* All members registered for iGEM Slack; David created internal communication group
* Joanna collected team questions for Wednesday 2 PM iGEM ambassador meeting
* Full team conducted transformation experiment with competent cells and ampicillin resistance plasmid
* Prepared PowerPoint and educational materials on micropipette usage and lab safety for Year 9 lesson
* Dry lab explored MATLAB and GitLab for modelling and wiki development
* Teams researched protease immobilization methods and types for Cp19k breakdown

✓ Completed

* Successfully carried out transformation protocol and plated cells
* Completed Slack onboarding for all members
* Finalized Year 9 educational presentation materials

→ Next Steps

* Assess transformation results (June 10/11) and calculate efficiency
* Deliver Year 9 lesson (June 10, Period 3: 11:15-12:15)
* Continue MATLAB/GitLab orientation

June 10

Logged by Zahara Greener

▼

👥 Members Present

**08:45 ~ 12:15:** Jayden Leung

**08:45 ~ 15:15:** Asger, JingJing, Zahara, William, Joanna, Lifu, Jackson, David

🔬 Activities

Dry Lab

* Mr. Edwards recommended CP-19k production modelling software
* Members explored proteins and modelling software
* Worked on building wiki

Wet Lab

* Checked competent bacteria efficiency
* Measured bacteria transformations
* Designed plasmid circuit
* Rehearsed presentation

✓ Completed

* Asger, Claire, David, Zahara delivered Year 9 presentation on micropipettes, lab safety, and iGEM overview

→ Next Steps

* Design GFP test circuit
* Select wells from distribution kit

June 11

Logged by Claire Park

▼

👥 Members Present

**11:15 ~ 12:45:** Jackson, David, Claire, Asger

**09:45 ~ 15:15:** Zahara, William, Joanna, Jayden Leung, Jayden Liu, Lifu, Evelyn

🔬 Activities

Wet Lab

* Asger and Jayden attempted GFP circuit on sbolcanvas.org (unsuccessful - contacted iGEM technicians)
* Asger built GFP circuit on Benchling using iGEM distribution kit sequence
* Claire, Yixuan, Zahara discussed experimental procedure outline
* Jayden Leung, Jayden Liu, Asger researched proteases for BiCp19k hydrolysis (522 bp, ~174 amino acids)
* Claire researched BiCp19k AlphaFold models on UniProt

Dry Lab

* Lifu, David, William, Jackson developed wiki Teams section with member profiles
* Joanna, Evelyn, JingJing created wiki animations (ships, fish, sharks)
* Refined team logo and drafted wiki layouts
* Started Contact section

✓ Completed

* Finished team member profiles (2/3 sub-tasks)
* Zoom meetings with iGEM ambassadors and ship captain
* Asger completed GFP circuit on Benchling

→ Next Steps

* Continue attribution section and lab logs
* Research protease active site structures

June 12

Logged by Claire Park

▼

👥 Members Present

**08:15 ~ 15:15:** Claire, Yixuan, Zahara, Asger, David, William, JingJing, Joanna, Lifu, Jayden Leung

🔬 Activities

Wet Lab

**Golden Gate Assembly:**

* Mr. Edwards presented golden gate assembly introduction
* Component distribution: Promoter (Asger), RBS (Claire), Plasmid (Zahara), Terminator (Jayden Leung), GFP (Yixuan)
* Asger, Zahara, Jayden Leung completed DNA transformation, incubated overnight
* Claire and Yixuan began transformation (heat shock and shaking stages)
* Asger identified two additional proteases
* Zahara identified proteases using FTmap
* All parts stored in school freezer

Dry Lab

* Joanna created cartoon images
* Lifu, David, William implemented animations using Sora
* Set up wiki subcategories per iGEM guides
* William learning MATLAB
* Joanna emailed about Year 6 teaching

Asger began Suzhou trip PowerPoint; Mr. Edwards organized trip logistics

✓ Completed

* Finished 'Team Members' wiki section

→ Next Steps

* Examine transformation efficiency, inoculate broth, mini-prep
* Upload lab logs to wiki

June 13

Logged by Claire Park

▼

👥 Members Present

**08:15 ~ 15:15:** Claire, Yixuan, Asger, David, William, JingJing, Joanna, Jayden Leung

🔬 Wet Lab Activities

**Transformation Results - Only controls worked:**

| DNA | Result | Control |
| --- | --- | --- |
| RBS (23O) | ✗ | ✗ |
| Plasmid (7G) | ✗ | ✗ |
| Promoter (15O) | ✗ | ✓ |
| Terminator (19E) | ✗ | ✓ |
| GFP (23G) | ✗ | ✓ |

**Analysis:** Methods verified (controls successful). Problem likely with resuspension or distribution kit.

**Troubleshooting Variables:**

| Sample | Water (µL) | Time (min) | Temp (°C) |
| --- | --- | --- | --- |
| K13 (Yixuan) | 5 | 5 | 37 |
| M21 (Claire) | 5 | 15 | 25 |
| A4 (Jayden) | 10 | 5 | 37 |
| I19 (Asger) | 10 | 15 | 25 |

Dry Lab

* JingJing and Joanna: Wiki animations, SnapGene logo, promotion poster, video planning
* William: MATLAB tutorials
* David: Social media accounts creation

✓ Completed

* Suzhou presentation finished
* Email setup: WCIS.Barnacure.iGEM@wellingtoncollege.cn

June 16

Logged by David

▼

👥 Members Present

**08:15 ~ 15:15:** David, Asger, Yixuan, Zahara, Lifu, William

🔬 Activities

Wet Lab

* Varied suspension parameters: time, temperature, plasmid concentration
* One transformation successful - breakthrough achieved!
* Yixuan contacted STU iGEM team and PhD student for protocol advice
* Mr. Edwards retried transformation and Golden Gate Assembly

Dry Lab

* William continued MATLAB tutorials
* David began wiki file imports

✓ Completed

* Colonies grew after longer incubation
* Switched framework to React
* Identified enzymes based on temperature/pH

→ Next Steps

* Troubleshoot distribution kit part lifting
* Determine optimal enzymes

June 17

Logged by David and Zahara

▼

👥 Members Present

**08:15 ~ 15:15:** David, Asger, Yixuan, Zahara, Lifu, William

🔬 Activities

Dry Lab

* William designed biofouling public questionnaire
* David and Lifu upgraded lab log, completed project description
* Converted TypeScript to HTML/CSS/JavaScript for GitLab

Wet Lab

* Yixuan and Asger transformed plasmids using two methods
* Developed wet lab workflow

✓ Completed

* Asger finalized protease order list
* Suzhou presentation finalized

→ Next Steps

* Understand protein isolation protocol
* Prepare biochemistry pathway presentation
* Research protease testing
* Focus on gold award requirements

June 23

Logged by Jayden Leung

▼

👥 Members Present

**09:00 ~ 16:00:** Yixuan, Asger, Zahara, Jayden Liu, Jayden Leung, Joanna, JingJing, William, David, Jackson, Evelyn

🔬 Activities

* Mr. Edwards presented on modelling genetic circuits
* JingJing researching circuit modelling

Dry Lab

* Jackson, Evelyn, JingJing refined video script
* William modeling CP-19k production in MATLAB
* Joanna created website animations

Wet Lab

* Practiced miniprep protocol (pET28A)
* Stored plasmids at -20°C
* Conducted electrophoresis trial
* Learned SnapGene gel simulation

✓ Completed

* Miniprep and electrophoresis trials
* Finalized video script

→ Next Steps

* Transform CP-19k tomorrow
* Film promotional video

June 24

Logged by Jayden Leung

▼

👥 Members Present

**09:00 ~ 16:00:** Yixuan, Asger, Zahara, Jayden Leung, Joanna, JingJing, William, David, Lifu, Evelyn

🔬 Activities

Dry Lab

* Made cardboard props and animations
* Started filming promotional video
* David and Lifu continued wiki
* William researching MATLAB modeling

Wet Lab

* Previous electrophoresis failed (low DNA concentration)
* Successfully transformed CP-19k plasmids

✓ Completed

* Filmed/edited first 30 seconds of video
* Completed CP-19k transformation and plating

→ Next Steps

* Continue filming
* Inoculate transformed bacteria

June 25

Logged by Jayden Leung

▼

👥 Members Present

**09:00 ~ 16:00:** Yixuan, Asger, Zahara, Jayden Leung, Jayden Liu, Joanna, JingJing, William, David, Lifu, Evelyn, Jackson

🔬 Activities

Dry Lab

* Video work: animations, music, editing
* Asger and David acted; others filmed
* William continued modeling research
* Jackson began public opinion survey

Wet Lab

* Observed significant bacterial growth
* Inoculated bacteria in LB medium
* SDS-PAGE trial (egg whites, fish, BSA)
* Lisa and Chloe visited: requested professional interviews and social media expansion

✓ Completed

* Completed SDS-PAGE trial

→ Next Steps

* Make glycerol stock
* STU trip survey
* Bradford assay trial

June 26

Logged by Jayden Leung

▼

👥 Members Present

**09:00 ~ 16:00:** Yixuan, Asger, Zahara, Jayden Leung, Jayden Liu, Joanna, JingJing, William, David, Lifu, Evelyn, Jackson

🔬 Activities

Dry Lab

* Evelyn: video editing (transitions, audio)
* Interactive website interface development
* Social media expansion; WeChat post created
* Mr. Edwards filmed drone waving video

Wet Lab

* LB medium: 4/5 trials successful
* Created 8 glycerol stocks (-80°C)
* Fixed SDS-PAGE (buffer issue)
* Successfully ran SDS-PAGE, dyed and de-stained gel

✓ Completed

* Fixed electrophoresis, prepared gel
* Made 8 glycerol stocks
* First WeChat post published

→ Next Steps

* Finalize Suzhou presentation
* Finalize STU questions
* Create summer workflow

June 27

Logged by Claire Park

▼

👥 Members Present

**09:00 ~ 16:00:** Yixuan, Asger, Zahara, Jayden Leung, Jayden Liu, Joanna, JingJing, William, David, Lifu, Evelyn, Jackson, Claire

🔬 Activities

Dry Lab

* David and Lifu: attributions with fluid effects
* Jackson: completed public engagement form
* Claire filmed for video
* Full Suzhou presentation rehearsal

Wet Lab

* Checked de-stained SDS-PAGE gel
* Followed gel drying protocol
* Photographed protein bands (blue light reader)
* Finalized post-summer workflow
* Claire completed Suzhou script

✓ Completed

* Finished SDS-PAGE, photographed bands
* Wet lab workflow finalized
* Suzhou rehearsal completed

→ Next Steps

* Return after summer for protease and kit work

### August 2025

Aug 28

Logged by Zahara Greener

▼

👥 Members Present

**13:45 ~ 14:15:** Zahara, Yixuan, Claire

🔬 Wet Lab Activities

* Discussed 3-week wet lab plans
* Reviewed CP-19k purification procedures
* Structured schedule (timing, equipment, assignments)

→ Next Steps

* Test CP-19k purity via SDS-PAGE
* Identify additional protease assays

Aug 29

Logged by Zahara Greener

▼

👥 Members Present

**13:45 ~ 14:15:** Zahara, Yixuan, Claire, Jayden Leung, Asger

🔬 Wet Lab Activities

* Thawed half of CP-19k glycerol stocks
* Inoculated in LB medium (1:10 ratio)
* Overnight incubation

→ Next Steps

* Induce CP-19k expression (IPTG) on Monday
* Purify CP-19k samples

### September 2025

Sept 2

Logged by Jayden Leung

▼

👥 Members Present

**15:45 ~ 17:30**

🔬 Wet Lab Activities

* Started nickel column protein purification
* Obtained pellets, resuspended with lysis buffer
* Isolated supernatant with target protein

Sept 4

Logged by Jayden Leung

▼

🔬 Wet Lab Activities

* **Claire and Yixuan:** Started 250mL inoculation for protein expression
* **Jayden Liu, Zahara, Jayden Leung:** Continued nickel column purification, prepared SDS-PAGE samples

Sept 5

Logged by Jayden Leung

▼

🔬 Wet Lab Activities

* **Claire and Yixuan:** Multiple centrifugation rounds for pellets
* **Zahara, Jayden Liu, Jayden Leung:** Ran SDS-PAGE with purified proteins
* Gel removed, awaiting Monday staining

Sept 8

Logged by Jayden Leung

▼

🔬 Wet Lab Activities

* Planned next steps to complete project
* Started SDS-PAGE staining process

Sept 9

Logged by Jayden Leung

▼

🔬 Wet Lab Activities

* Started nickel column for 250mL culture
* Resuspended pellets in lysis buffer, centrifuged
* Protein isolated in supernatant
* SDS-PAGE results: bands faint and unclear

Sept 12

Logged by Claire Park

▼

🔬 Wet Lab Activities

* Completed nickel column purification for 250mL culture

Sept 15

Logged by Claire Park

▼

🔬 Wet Lab Activities

* Continued SDS-PAGE on CP-19k culture
* Calibrated Bradford assay for CP-19k quantification
* Excellent calibration curve: r = 0.99

Sept 16

Logged by Claire Park

▼

🔬 Wet Lab Activities

* Performed Bradford assay on CP-19k samples
* Determined CP-19k protein concentration

Sept 17

Logged by Claire Park

▼

🔬 Wet Lab Activities

Protease Testing

**Trypsin Preparation:**

* Prepared 50mM ethanoic acid
* Measured 0.004g trypsin powder, mixed
* Combined 50µL trypsin with 1000µL CP-19k (1:20 ratio)
* Incubated at 37°C overnight

**Alcalase Preparation:**

* Prepared 10mM Tris-HCl buffer
* Measured 0.11g alcalase powder (7-15 U/mg; 1.1mg/mL)
* Combined 50µL alcalase with 1000µL CP-19k
* Incubated at 37°C overnight

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# Engineering Success

#### Contents

* [Overview](#overview)
* [Design Phase](#design)
* [Build Phase](#build)
* [Test Phase](#test)
* [Learn Phase](#learn)
* [Iteration](#iteration)

## Engineering Cycle Overview

---

Our engineering cycle focused on the synthesisation of barnacle cement's key structural protein Balcp19K (abbreviated to CP19K). CP19K plays a crucial role in underwater adhesion. The objective of this cycle was to create a plasmid sequence to be expressed within a bacterial host capable of CP19K synthesisation in the laboratory. This synthetic production enables testing of different proteases for cement protein degradation which can then be used in antifouling products such as biofilms, marine coatings or cleaning products.

## Design Phase

---

Following the Design phase of the engineering cycle, two promoters (T7 and J23119) and two regulatory operators (LacO and TetO) were selected to form four promoter–operator combinations. These were chosen for their wide range of use. The goal at this stage was to identify which configuration could potentially achieve high protein yield.

#### Design Components Selected

* **Promoters:** T7 and J23119
* **Operators:** LacO and TetO
* **Combinations:** Four promoter-operator configurations
* **Goal:** Identify optimal configuration for high CP19K yield

## Build Phase

---

The Build stage involved constructing a mechanistic model of CP19K expression under each promoter–operator combination using a system of coupled ordinary differential equations. These equations captured transcription, translation, and degradation processes, as well as repressor–inducer binding and ATP consumption, parameterised from literature data. Each system variant was simulated for 12 000 minutes in MATLAB to predict relative mRNA and protein output.

#### Mechanistic Model Components

* **Mathematical Framework:** Coupled ordinary differential equations
* **Biological Processes:** Transcription, translation, and degradation
* **Regulatory Elements:** Repressor-inducer binding dynamics
* **Resource Management:** ATP consumption modeling
* **Simulation Platform:** MATLAB with 12,000 minute timeframe

## Test Phase

---

During the Test and Learn phases, simulation results revealed that both T7-based systems outperformed J23119-based ones, producing approximately twice the amount of CP19K over the simulated period. This is matches with known biological characteristics of the 2 promoters and operators, as T7 is a strong bacteriophage promoter driven by T7 RNA polymerase, known for its high transcription rate. LacO marginally outperformed TetO by around 3 %, indicating slightly tighter repression control.

#### Key Test Results

* **T7 vs J23119:** T7-based systems produced ~2x more CP19K
* **LacO vs TetO:** LacO outperformed TetO by ~3%
* **Optimal Configuration:** T7-LacO combination
* **Biological Validation:** Results align with known promoter characteristics

## Learn Phase

---

Overall, this engineering cycle demonstrated that the T7–LacO construct is most efficient for CP19K synthesis, also highlighting the effectiveness of computational modelling as a predictive and cost-efficient tool in synthetic biology. These results can then be used in the to test different proteases and identifying the most efficient CP19K-degrading enzyme.

#### Key Learnings

* **Optimal Design:** T7-LacO construct identified as most efficient
* **Methodology Validation:** Computational modeling proven effective
* **Cost Efficiency:** Predictive modeling reduces experimental costs
* **Next Steps:** Results inform protease testing strategy

## Ni-NTA Purification Optimization

---

### Design

To run the Ni-NTA and purify the protein after induced expression, we needed to calculate how much glycerol stock we should revive and how much broth to make. We calculated the optimum volume of broth to make, accounting for how much of our glycerol stock we were using and the total volume our centrifuge (Bioridge TGL-16.5M) could centrifuge at once. From these considerations, we decided 40ml of broth would be optimum to centrifuge.

### Build

Following the protocol, we had discarded the supernatant and resuspended the cell pellets in lysis buffer. We then incubated, discarded the pellets and underwent the Ni-NTA purification process. We took 10ul aliquots every stage and added 5ul of 4x SDS-page loading buffer.

### Test

Our SDS-page testing the protein purification revealed no bands. We suspected that the aliquots we had taken were too small and that the protein concentration was too little for visible bands to show up on the SDS-page

### Learn

We revived a larger amount of glycerol stock, created 250ml of broth and took larger aliquots of 40ul.

#### Iteration Summary

* **Issue Identified:** Insufficient protein concentration for detection
* **Root Cause:** Small aliquot volumes and limited starting material
* **Solution Implemented:** Increased broth volume and aliquot sizes
* **Scale-up:** 40ml → 250ml broth, 10μl → 40μl aliquots

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# Experiments & Protocols

Comprehensive, step-by-step protocols for all experiments conducted in the Barnacure project, designed to provide sufficient information for other teams to replicate our work.

#### Protocols

* [Justification & Tips](#justification)
* [Part 1: Transformation](#transformation)
* [Part 2: Inoculation](#inoculation)
* [Part 3: Protein Expression](#expression)
* [Part 4: Glycerol Stocks](#glycerol)
* [Part 5: Ni-NTA Purification](#purification)
* [Part 6: Protein Digest](#digest)
* [Part 7: SDS-PAGE](#sds-page)

## Justification of procedures:

---

To test the protease efficacy, we chose to run an SDS-page as we did not have access to a nanodrop. We ran a trial Bradford assay to see protein concentration, and we recognised that it could not provide quantitative data on molecular weight of proteins.

We chose to use the Ni-NTA to purify the proteins as it would yield a pure sample that can be further tested on and is within budget. To accommodate for this procedure, we included a 6-His tag in the protein that will bind to the Ni ions.

### Tips and tricks:

* With SDS-pages, make sure there is enough running buffer as it had short-circuited the machine
* Be careful when loading the protein samples as if done too quickly, it may splash on to surrounding wells, obscuring results.
* The SDS-pages need to be destained until most of the gel is clear to ensure best photo clarity when analysing.
* When heating water baths up to 95ºC, it is more efficient to boil water and pour it into the water bath
* Pay attention to any guides provided by the manufacturer, especially the units of proteases
* If it is not possible to destain a gel immediately using the destaining solution, leave the gel in distilled water to slowly destain overnight
* The density of the sample should be greater than the density of the solution in the well

## Part 1: Transformation of PET21a+balcp19k plasmids into BL21(DE3) competent cells

---

![Agar Plate Results](https://static.igem.wiki/teams/5682/experiments/exp-agar.webp)

1. Transformation of Recombinant Plasmid into Expression Host
2. Take competent E. coli BL21(qDE3) cells from a -70 °C freezer and thaw them on ice.
3. Add 1 μL of pET21a-balcp19k, to 50 μL of competent E. coli BL21(DE3) cells. Incubate on ice for 30 minutes.
4. Heat shock at 42 °C for 60 seconds, then return to ice for 5 minutes.
5. Add 950 μL of antibiotic-free LB liquid medium (or SOC) to the mixture and shake at 220rpm/min at 37ºC for 1 hour.
6. Centrifuge the cultured cells at 6800g for 3 minutes. Discard 800ul of the supernatant using a pipette tip and resuspend the cells in the remaining supernatant.
7. Plate 100 μL of the resuspended mixture onto LB agar plates containing ampicillin. After the liquid is fully absorbed, invert the plates and incubate at 37 °C overnight.

## Part 2: Inoculating a Liquid Bacterial Culture

---

![Inoculation Setup](https://static.igem.wiki/teams/5682/experiments/exp-innoc.webp)

![Culture Growth](https://static.igem.wiki/teams/5682/experiments/exp-inoc2.webp)

1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:4 g NaCl, 4 g Tryptone, 2 g Yeast Extract, and dH2O to 400 mL

Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.

2. When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration.

[add exp table here]

Note: If you intend to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2L Erlenmeyer flask.

3. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
4. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
5. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
6. Incubate bacterial culture at 37°C 250rpm for 12-18 hr in a shaking incubator

*(From: https://www.addgene.org/protocols/inoculate-bacterial-culture)*

## Part 3: Induced Expression of the Cp19k Protein

---

8. The next day, dilute the overnight culture 1:100 into 5 mL of fresh LB medium containing ampicillin. Incubate at 37 °C, shaking at 220 rpm/min for 1.5 hours. (Measure OD at 600 nm. When OD₆₀₀ reaches between 0.4–0.6,) add IPTG (1.0mM) to a final concentration of 1.0 mM to induce recombinant protein expression.
9. After induction at 27 °C for 4 hours, take 1.5 mL of culture. Centrifuge at 10,000 rpm/min for 5 minutes. Discard the supernatant and resuspend the pellet in 400 μL PBS.

## Part 4: Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids

---

1. After you have bacterial growth, add 600 μL of the overnight culture to 400 μL of 50% glycerol in a 1.5ml centrifuge tube.
2. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Avoid freeze and thaw cycles as it reduces shelf life.
3. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
4. Grow your bacteria overnight at the appropriate temperature. Growth conditions, including copy number and growth temperature, can be found on your plasmid's information page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.

*(From: https://www.addgene.org/protocols/create-glycerol-stock)*

## Part 5: Extraction and purification of proteins via Ni-NTA column (Purification of 6xHis-tagged Proteins Under Native Conditions)

---

![Ni-NTA Column Purification](https://static.igem.wiki/teams/5682/experiments/exp-nta.webp)

Before use, native Lysis Buffer must be supplemented with lysozyme and BenzonaseR Nuclease. Dissolve the contents of the lysozyme vial in 600 μl of native Lysis Buffer. Add 100 μl of the lysozyme solution to a 10 ml aliquot of native Lysis Buffer. Store the remaining lysozyme solution at -20°C. Thaw the vial containing Benzonase® Nuclease solution and add 10 μl to the 10 ml aliquot of native Lysis Buffer.

### Materials:

* E. coli cell pellet from 250 ml culture
* 2x SDS-PAGE sample buffer

### Procedure

1. Thaw the cell pellet for 15 min on ice and resuspend the cells in 10 ml native Lysis Buffer. Ensure that lysozyme and Benzonase® Nuclease have been added to the lysis buffer.
2. Incubate on ice for 30 min. Mix 2-3 times by gently swirling the cell suspension.
3. Centrifuge lysate at 14,000 x g for 30 min at 4°C to pellet the cellular debris. Retain the cell lysate supernatant. The supernatant contains the soluble fraction of the recombinant protein.
4. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the supernatant and store at -20°C for SDS-PAGE analysis.
5. Gently resuspend the resin in a Fast Start Column by inverting it several times.
6. Break the seal at the outlet of the column, open the screw cap, and allow the storage buffer to drain out. It is important that the outlet seal is broken before the screw cap is removed.
7. Apply the cell lysate supernatant from step 3 to the column.
8. Collect the flow-through fraction. Add 5 μl 2 x SDS-PAGE sample buffer to a 5 μl aliquot of the flow-through fraction and store at -20°C for SDS-PAGE analysis.
9. Wash the column 2 times with 4 ml of native Wash Buffer. Collect both wash fractions. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of each wash fraction and store at -20°C for SDS-PAGE analysis.
10. Elute bound 6xHis-tagged protein with two 1 ml aliquots of Native Elution Buffer.
11. Collect each elution fraction in a separate tube. Add 5 μl 2x SDS- PAGE sample buffer to a 5 μl aliquot of each elution fraction and store at -20°C for SDS-PAGE analysis.
12. Analyze all fractions by SDS-PAGE.

- approx. 25mg/L yield

*(From: EN-QIAexpress-Ni-NTA-Fast-Start-Handbook)*

## Part 6: Protein Digest for native proteins

---

1. Reconstitute lyophilized powder in suitable/recommended solvent to form a concentration of 50mM. For Trypsin, use acetic acid, for alcalase, use Tris HCl with pH range between 7-9. Adjust volume of reconstituted solution to account for protease:protein (unit/mg) ratio.
2. Incubate at 37ºC from 4 hours to overnight
3. Take an aliquot and add SDS-page 4x loading buffer
4. Heat shock for 10 minutes at 95ºC

*(Adapted from https://www.upstate.edu/proteomics/prep/trypsin.php)*

## Part 7: SDS-PAGE to analyse the results of enzyme digest

---

![SDS-PAGE Setup](https://static.igem.wiki/teams/5682/experiments/exp-sds.webp)

![SDS-PAGE Results](https://static.igem.wiki/teams/5682/experiments/exp-sds2.webp)

### Materials:

* SDS-PAGE gel (e.g., 12% resolving, 4% stacking)
* Protein samples
* 4x Loading buffer
* 95 ºC water bath
* Protein ladder (molecular weight marker)
* Electrophoresis tank and power supply
* Running buffer (1× Tris-Glycine-SDS)
* Micropipettes and tips

### 1. Prepare Samples

1. Mix protein sample (e.g., lysate, supernatant, pellet) with SDS-PAGE loading buffer in the ratio of 3:1
2. Heat all samples at 95°C for 5 minutes to denature proteins and ensure uniform charge.
3. Briefly spin down to collect condensation.

### 2. Set Up Gel

Insert SDS-PAGE gel into electrophoresis tank.

Fill inner and outer chambers with 1× running buffer (3:1, water: buffer).

### 3. Load Samples

1. Load 40μL of protein ladder in the first and/or last lane.
2. Load 40μL of sample (with loading buffer) per lane (depending on well size).

### 4. Run the Gel

1. Connect electrodes: black (–) to top, red (+) to bottom.
2. Run at 120 V through the resolving gel until the dye front is near the bottom, make sure to not let the dye front run through. (~45–60 minutes).

### 5. Post-Electrophoresis

Carefully open the seal on the inner chamber of the SDS-page machine

Remove the gel (sandwiched in between two glass plates)

Use a spatula to carefully pry open the glass and remove the gel

Place the gel in

Rinse the gel with distilled water twice, carefully pouring out the water afterwards

Add Coomassie Brilliant Blue stain until it just covers the gel.

Shake for 1-2 hours

Pour out the stain and recycle it

Add destaining solution until it just covers the gel

Shake for 30-90 mins

Repeat previous 2 steps with new destaining solution until the bands are visible (leave overnight for better effects).

### Notes

Use fresh gels for clearer resolution.

12% resolving gel is suitable for proteins ~20–100 kDa.

Keep your protein ladder visible to estimate molecular weights. It is recommended to add the standard to the 1st and last well of the gel to account for uneven dye fronts.

Run multiple sample types (lysate, supernatant, pellet) to check protein solubility.

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# Results

Comprehensive experimental results from plasmid design through protease digest analysis, demonstrating our journey in developing an effective barnacle cement protein degradation system.

#### Contents

* [Plasmid Design](#plasmid-design)
* [Transformation](#transformation)
* [Inoculation](#inoculation)
* [Glycerol Stock](#glycerol-stock)
* [Re-growth](#regrowth)
* [IPTG Induction](#iptg-induction)
* [Ni-NTA Purification](#purification)
* [SDS-PAGE Analysis](#sds-analysis)
* [Protease Digest](#protease-digest)

## 1. Plasmid Design

---

For our plasmid design, we chose pET-21a(+) due to its strong expression ability in E. coli. It consisted of:

#### Key Plasmid Components

* **Ampicillin Resistance (ampR):** This allowed us to select for transformed E. coli cells by growing them in ampicillin.
* **LacI Repressor:** We included LacI to control gene expression. LacI binds to the lac operator, preventing transcription until we induce it with IPTG.
* **Lac Operator:** The lac operator ensures tight regulation by blocking transcription in the absence of IPTG. IPTG deactivates LacI, allowing expression to occur.
* **Ribosome Binding Site (RBS):** The RBS ensures efficient translation of the gene, increasing protein yield after induction.
* **LacI Promoter:** The lacI promoter drives the expression of the LacI repressor, maintaining control over when the gene is expressed.
* **Origin of Replication (ori):** The pBR322 ori allows high-copy replication of the plasmid in E. coli, ensuring enough plasmid and protein are produced.
* **Balcp19k gene:** We inserted our target gene in the MCS.

Overall, this design gives us precise control over protein expression, maximizing yield and minimizing cell stress.

### Sequence Optimization

For the cp19k coding sequence, we originally found a biobrick on the iGEM registry. However, we noticed that its CG content was not within the optimal range of 40-60% for best expression in E. coli. To combat this, we researched literature and found an optimized sequence in Xu ZhenZhen, 2020, which optimized CG content to 50%, the resulting gene a total of 522bp. Then we added a 6xHis tag and a start and stop codon to make a coding sequence of 549 bp, which we named Balcp19k and submitted to the registry as part BBa\_25FRPPH9. This was sent to the company Nanjing Genscript where the plasmid was sequenced and mailed back to our school.

## 2. Transformation of Plasmid

---

Following the protocol from the iGEM website with modifications (see Experiments, Part 1), we transformed the sequenced plasmids to BL21 (DE3) competent cells.

Then, we spread the transformed bacteria on agar plates with AmpR.

![Transformation Results](https://static.igem.wiki/teams/5682/results/results1.avif)

The transformation of the plasmid was successful and we had bacterial growth on agar plates with Ampicillin resistance (top plate) and control (bottom right). For the inoculation, we extracted the individual colonies marked with green on the top plate.

## 3. Inoculation

---

![Inoculation Results](https://static.igem.wiki/teams/5682/results/results2.avif)

We inoculated (following the protocol in Experiments, Part 2) the transformed cells in LB medium and left them overnight in a shaking incubator at 37 degrees celsius, 250rpm. The bacteria had successfully grown in 4 of the 5 tubes (tube 1, 3, 4, 5 in the image above), as evidenced by the turbidity/cloudiness of the solution.

## 4. Glycerol Stock Storage

---

Following our successful inoculation, due to our limited time frame, we had to halt the experiments over the summer and prepare the glycerol stocks for long term storage. We combined 600 microliters of the overnight bacterial culture with 400 microliters of 50% glycerol solution in cryotubes. These stocks were stored at –80 degrees celsius, allowing us to preserve our stock over the summer holiday.

## 5. Re-growth of Bacteria (After the Summer)

---

After the summer break, we revived our transformed cells and retrieved the glycerol stocks from the freezer where we thawed them. A small sample was used to inoculate fresh LB medium containing ampicillin which was then stored overnight in a shaking incubator at 37 degrees celsius. This successfully regrew the bacteria and provided the needed active bacterial culture needed for the next steps of protein expression.

## 6. Induced Expression with IPTG

---

After confirming successful transformation and regrowth of our engineered E. coli BL21 (DE3) cells containing the pET-21a (+)-Balcp19k plasmid, we proceeded to induce protein expression of the cp19k gene using IPTG as the inducer under the T7 promoter system.

### Procedure

We first diluted overnight cultures 1:100 into 5 mL of fresh LB medium containing ampicillin (100 µg/mL) to maintain plasmid selection. The cultures were incubated at 37 °C with shaking at 220 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.6, corresponding to the mid-logarithmic growth phase of the optimal point for protein induction. We used a spectrophotometer to measure OD₆₀₀ value of 0.61, indicating healthy bacterial growth and a suitable cell density for induction.

At this point, IPTG was added to a final concentration of 1.0 mM to initiate transcription from the T7 promoter by inactivating the LacI repressor. The culture was then transferred to 27 °C and incubated for 4 hours with shaking at 220 rpm to promote soluble protein expression and reduce inclusion body formation, which can occur at higher temperatures.

After induction, 1.5 mL of the culture was harvested by centrifugation at 10,000 rpm for 5 minutes. The supernatant was safely discarded, and the resulting cell pellet was resuspended in 400 µL PBS for downstream protein analysis.

#### Key Results

This step successfully marked the induced expression phase of our cp19k protein. The recorded OD and pellet formation confirmed healthy post-induction growth, suggesting that protein expression occurred as expected. The samples were subsequently prepared for Ni-NTA purification and SDS-PAGE analysis, described in the following section.

## 7. Purification with Ni-NTA Column

---

After successfully inducing expression, protein should be in the cell pellet. The cell pellet is then resuspended in Lysis buffer. This is incubated on ice for 30 minutes and centrifuged at 14,000x for 30 minutes at 4 degrees. The resulting supernatant will contain the soluble cement protein. A 5µL aliquot was taken and added with 5µL of SDS page buffer, to run a trial of SDS page, confirming whether the protein was present. In the first trial, there were no visible bands, presenting a lack of protein. We hypothesized the reason being that a low volume of E. coli cell pellet was used so another 250ml inoculation was started in an attempt to obtain more protein.

### Second Trial - Improved Protocol

The 250ml inoculation was centrifuged to extract the E. coli pellets and resuspended in lysis buffer for the second trial of protein purification. The supernatant containing the protein is filtered through a Fast start column and the flow through is collected as the first fraction. The column is further washed twice with 4ml Wash buffer and the flow through was collected again for the 2nd and 3rd fraction. The column is washed twice more with 1ml Native elution buffer to form the 4th and 5th fraction.

### Bradford Assay Results

The concentration of protein of each fraction is analyzed using the Bradford Assay, where all fractions yielded proteins, presenting success. A calibration curve was created with increasing concentration of protein, so the absorbance of each fractions can be used calculate the concentration of protein in them. The fraction with the most protein was the 2nd elution wash (5th fraction) with 0.2mg/L. So this fraction was decided to be used for the enzyme protein digest, as it contained the most protein.

![Bradford Assay Results](https://static.igem.wiki/teams/5682/results/results3.avif)

## 7. SDS-PAGE Analysis

---

The amount of protein in each fraction is analyzed further by running an SDS page.

![SDS-PAGE Results](https://static.igem.wiki/teams/5682/results/results4.avif)

The second trial showed visible bands which is promising, showing that protein was present. It confirms the error in the first trial where total E. coli pellet used was too small to produce enough protein. The SDS page was "smiling" so we had adjusted our technique using a more appropriate voltage in following gels and the issue was resolved.

## 8. Protease Digest

---

![Protease Digest Setup](https://static.igem.wiki/teams/5682/results/results5.avif)

After the purification of CP-19k using the Ni-NTA, individual samples were incubated with proteases alcalase and trypsin at 37ºC overnight to test their respective efficacies.

### Analysis of Purification Results

The final fraction of the Ni-NTA (elution 2) yielded a moderately impure sample, with 10 bands at different molecular weights. This suggests the solution was contaminated with co-eluted proteins, indicating insufficient washes during the purification process ([GoldBio](https://goldbio.com/documents/3961/Affinity%20His-Tag%20Purification%20utilizing%20Nickel%20NTA%20Magnetic%20Agarose%20Beads.pdf)). The most prominent bands had molecular weights of 35.4, 26.9 and 13.4 kDa, rather than the anticipated 19kDa. This may have been because SDS pages utilise total protein charge as a migration mechanism, meaning that it is not highly sensitive to molecular weight, but rather the charge of SDS-bound residues. Disordered proteins, or proteins that have undergone glycosylation, methylation can all alter the presented molecular weight. ([Nature](https://www.nature.com/articles/srep13370)) ([PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7177777))

However, this discrepancy could also be caused by the presence of formed disulphide bonds which may have been prevented by the addition of ß-mercaptoethanol. ([Novagen](https://wolfson.huji.ac.il/purification/PDF/Protein_Refolding/NOVAGEN_NiNTA_purification_resins.pdf))

### Alcalase Results

![Alcalase Digest Results](https://static.igem.wiki/teams/5682/results/results6.avif)

The two alcalase samples varying results. The first alcalase sample had a band at an initial 36.2 kDa as well at lower molecular weights of 17.2 and 13.1 kDa. This meant that the alcalase had cleaved the original protein into significantly smaller fragments. There appear to be no bands above 36.2 kDa meaning that the protein concentration of the sample was either too small or the fragment concentrations were too low to be visible. The second sample had yielded a blank lane, which could mean that the final aliquot collected had a loading sample with too low of a protein concentration.

### Trypsin Results

![Trypsin Digest Results 1](https://static.igem.wiki/teams/5682/results/results7.avif)

![Trypsin Digest Results 2](https://static.igem.wiki/teams/5682/results/results8.avif)

Trypsin was able to digest the prominent 35 kDa band and higher molecular weight bands, creating a secondary band at lower molecular weights at 31.6 kDa in both samples. This suggests that the enzyme was able to catabolise the protein, but not fully as there were still bands at 34.7 and 35.6 kDa. This proves that the protease has disputable efficacy but is unlikely to have targeted the desired protein.

### Discussion and Future Directions

#### Analysis of Ambiguities

These ambiguities suggest there were human errors (e.g. problems with loading the wells), unsuitable method choice or, measurement error, and an inadequate sample size. The methodology also could not provide the definite uncertainty of each band's molecular weight as it is relative to the protein ladder standard and is reliant on the clarity of the original .TIF file.

#### Recommendations for Future Work

An ideal cp-19k-ase would have a high affinity and specificity and could potentially be modelled off of certain areas of alcalase's active site. This confirms the hypothesised efficacy of proteases and cleavage sites in silico. If these experiments were to be repeated, a purer starting sample should be used, and another method of quantifying protease catabolism efficacy should be considered (e.g. thin layer chromatography, mass spectrometry and UV/Vis spectroscopy) ([ScienceDirect](https://www.sciencedirect.com/science/article/pii/S1567539424001373)) ([ACS Omega](https://pubs.acs.org/doi/10.1021/acsomega.0c05192))

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