iGEM 2020 — Team HKUST

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:HKUST/Notebook

iGEM HKUST

[ ]

[![](https://static.igem.org/mediawiki/2020/c/c5/T--HKUST--logo.png)](https://2020.igem.org/Team%3AHKUST)

* Project

  + [Description](https://2020.igem.org/Team%3AHKUST/Description)
  + [Design](https://2020.igem.org/Team%3AHKUST/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AHKUST/Contribution)
  + [Improvements](https://2020.igem.org/Team%3AHKUST/Improvements)
  + [Safety](https://2020.igem.org/Team%3AHKUST/Safety)
* [Dry Lab](https://2020.igem.org/Team%3AHKUST/Model)

  + [Protein Structure Pretiction](https://2020.igem.org/Team%3AHKUST/Model#heading-2)
  + [Molecular Dynamics Simulation](https://2020.igem.org/Team%3AHKUST/Model#heading-3)
  + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Model#heading-4)

  * [Wet Lab](https://2020.igem.org/Team%3AHKUST/Experiments)

    + [Description](https://2020.igem.org/Team%3AHKUST/Experiments#heading-1)
    + [Cloning](https://2020.igem.org/Team%3AHKUST/Experiments#heading-2)
    + [Expression & Purification](https://2020.igem.org/Team%3AHKUST/Experiments#heading-3)
    + [Thread Formation](https://2020.igem.org/Team%3AHKUST/Experiments#heading-4)
    + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-5)
    + [Draw-Processing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-6)
  * Human Practice

    + [Implementation](https://2020.igem.org/Team%3AHKUST/Implementation)
    + [Integrated Human Practice](https://2020.igem.org/Team%3AHKUST/Human_Practices)
    + [Collaborations](https://2020.igem.org/Team%3AHKUST/Collaborations)
    + [Education](https://2020.igem.org/Team%3AHKUST/Education)
    + [Partnership](https://2020.igem.org/Team%3AHKUST/Partnership)
    + [Entrepreneurship](https://2020.igem.org/Team%3AHKUST/Entrepreneurship)
  * Team

    + [Team Members](https://2020.igem.org/Team%3AHKUST/Team)
    + [Attributions](https://2020.igem.org/Team%3AHKUST/Attributions)
  * Parts

    + [Parts Overview](https://2020.igem.org/Team%3AHKUST/Parts)
    - [Basic Parts](https://2020.igem.org/Team%3AHKUST/Basic_Parts)
    - [Composite Parts](https://2020.igem.org/Team%3AHKUST/Composite_Parts)
  * Awards

    + [Award Criteria](https://2020.igem.org/Team%3AHKUST/Award)
    + [Achievements](https://2020.igem.org/Team%3AHKUST/Achievements)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:HKUST/Engineering

iGEM HKUST

[ ]

[![](https://static.igem.org/mediawiki/2020/c/c5/T--HKUST--logo.png)](https://2020.igem.org/Team%3AHKUST)

* Project

  + [Description](https://2020.igem.org/Team%3AHKUST/Description)
  + [Design](https://2020.igem.org/Team%3AHKUST/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AHKUST/Contribution)
  + [Improvements](https://2020.igem.org/Team%3AHKUST/Improvements)
  + [Safety](https://2020.igem.org/Team%3AHKUST/Safety)
* [Dry Lab](https://2020.igem.org/Team%3AHKUST/Model)

  + [Protein Structure Pretiction](https://2020.igem.org/Team%3AHKUST/Model#heading-2)
  + [Molecular Dynamics Simulation](https://2020.igem.org/Team%3AHKUST/Model#heading-3)
  + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Model#heading-4)

  * [Wet Lab](https://2020.igem.org/Team%3AHKUST/Experiments)

    + [Description](https://2020.igem.org/Team%3AHKUST/Experiments#heading-1)
    + [Cloning](https://2020.igem.org/Team%3AHKUST/Experiments#heading-2)
    + [Expression & Purification](https://2020.igem.org/Team%3AHKUST/Experiments#heading-3)
    + [Thread Formation](https://2020.igem.org/Team%3AHKUST/Experiments#heading-4)
    + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-5)
    + [Draw-Processing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-6)
  * Human Practice

    + [Implementation](https://2020.igem.org/Team%3AHKUST/Implementation)
    + [Integrated Human Practice](https://2020.igem.org/Team%3AHKUST/Human_Practices)
    + [Collaborations](https://2020.igem.org/Team%3AHKUST/Collaborations)
    + [Education](https://2020.igem.org/Team%3AHKUST/Education)
    + [Partnership](https://2020.igem.org/Team%3AHKUST/Partnership)
    + [Entrepreneurship](https://2020.igem.org/Team%3AHKUST/Entrepreneurship)
  * Team

    + [Team Members](https://2020.igem.org/Team%3AHKUST/Team)
    + [Attributions](https://2020.igem.org/Team%3AHKUST/Attributions)
  * Parts

    + [Parts Overview](https://2020.igem.org/Team%3AHKUST/Parts)
    - [Basic Parts](https://2020.igem.org/Team%3AHKUST/Basic_Parts)
    - [Composite Parts](https://2020.igem.org/Team%3AHKUST/Composite_Parts)
  * Awards

    + [Award Criteria](https://2020.igem.org/Team%3AHKUST/Award)
    + [Achievements](https://2020.igem.org/Team%3AHKUST/Achievements)

# Design

* [## Background](#heading-1)
* [## Project Goals/Targets/Principles](#heading-2)
* [## Project Design Overview](#heading-3)
* [## Timeline](#heading-4)
* [## Detailed Project Design](#heading-5)

# Background

***What is hagfish slime intermediate filament?***

Intermediate filament, with its protein nature and orientation, usually plays an important role in cytoskeleton for maintenance in the shape of the cells. However, in hagfish slime intermediate filaments, they are secreted out to the extracellular environment. Hagfish slime intermediate filament (IF) is originated from hagfish slime, together with mucin vesicles, hagfish IF forms a slime mass which entrains a large volume of sea water. Upon predation, hagfish could release large amounts of slime to clog the gills of predators as a defence mechanism [1]. There are several kinds of species of hagfish. Throughout our project, we focus on the IF from Pacific hagfish (Eptatretus stoutii).

Hagfish slime threads are composed of bundles of IF. IF consists of 2 subunits, alpha (ɑ) and gamma (ɣ) keratin filament. Both subunits possess similar molecular weight and size. ɑ-keratin filament and ɣ-keratin filament will undergo self-assembly to form a ɑ-helical coiled-coil protein structure. This form of IF is very soft and highly extensible, but has little stiffness [2].

And upon draw-processing (pulling the fibers with a certain strain), the ɑ-helical coiled-coil structure will break open and combine with neighbouring strands. This transits the coiled-coil structure into a structure called β-sheet. And it is this transition that grants the fibres with ultra-high stiffness which is comparable to spider dragline silk.

***Why hagfish slime intermediate filament?***

In our search for possible candidates as a textile fiber material, we compared their extensiveness, tensile strength, the ease of production in a bacterial chassis, and other unique characteristics.

Drawn hagfish slime threads’ textile strength is comparable to spider dragline silk, and its high extensibility makes it a strong candidate.

Spider dragline silk and cocoon silk exhibit impressive physical performance in a dry environment. However, they undergo a unique process, supercontraction, in wet state, in which the fibers will shrink and contract in length. The contraction of spider silk could lead to a 1000-fold decrease (~10Gpa to ~10MPa) in stiffness, and this is less desirable for a textile product. Hagfish slime thread experiences a similar contraction process. However it could be mitigated by extending the draw processing time or by chemical cross-linking [2].

For decades, spider silks have been facing a fundamental challenge in recombinant silk production. The large and repetitive spider silk protein genes are unfavourable to be expressed in bacteria or yeast. Last year’s high school grand prize winner GreatBay\_SZ successfully expressed recombinant spider silk using 2 repetitive sequences only. This greatly improved the possibility for spider silk production in bacterial chassis. However, there is yet insufficient evidence supporting that the recombinant spider silk with only 2 repetitive sequences could improve or retain its high material performance. We wanted to avoid changing the amino acids sequence while trying to retain the physical properties of the fibers.

Hagfish slime IF, with the ability to assemble autonomously while increment to stiffness is possible by draw-processing and chemical cross-linking, possess small in size and can be easily synthesised and with the stability to withstand unstable sea water conditions, make it our target candidate as a potential material for textile use.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | **Textile strength;** **Young’s Modulus (GPa)** | **Extensibility;** **Breaking strain** | **Ease of production in a bacterial chassis** | **Unique characteristics** |
| **Drawn hagfish slime threads** | ~1.1 [2] | ~36% [2] | / | Supercontraction in wet state, which leads to a decrease in stiffness. Could be mitigated by increasing drawing time and chemical cross-link |
| **Spider dragline silk** | ~1.2 | ~26% [3] | Inefficient production due to large and repetitive sequence | Supercontraction in wet state, which leads to a decrease in stiffness |
| **Cocoon silk (species** ***Bombyx mori***) | ~0.5 [4] | ~20% [5] | / | Supercontraction in wet state, which leads to a decrease in stiffness |

*Comparison of the key properties of biological fibers.*

***What are chromoproteins?***

Chromoproteins are a class of proteins that can give out colour. Unlike fluorescent proteins which absorb UV-light, chromoproteins absorb light in the visible region and emit a specific range of visible light only, which could be observed with the naked eye.

***Why chromoproteins?***

Chromoproteins are encoded by a single gene only, this allows protein expression to be relatively easy compared to engineering the entire complex metabolic pathway of organic dyes. This simpler dyeing method introduces less variables to the experiments, and results in greater reproducibility of experiments, as well as easier analysis of the results. In natural dyes, dyeing instability due to color fading may occur [6]. Chromoproteins are less well characterised in this aspect, therefore, we will try to determine whether chromoproteins face the same problem as natural dye.

# Project Goals / Targets / Principles

Inspired by the 2014 UCC team who attempted to produce recombinant hagfish intermediate filament (IF), we realized there is great potential in hagfish filaments. That’s why we propose a mass production platform for recombinant hagfish IF. We want to provide the foundational basis for turning it into a viable textile material.

While researching solutions to the water pollution problems caused by textile microfibres and the dyeing industry, we realized that as iGEM participants and as scientists in training, we must focus on sustainable development and how our actions will impact the society and environment for subsequent generations.

Therefore, our major goals for this project can be summarized as the following 3 elements of sustainability:

|  |  |  |
| --- | --- | --- |
| **1. Environmental** | **2. Economic** | **3. Social** |
| Sought to solve environmental problems caused by microfibre & dyeing using biological materials to reduce our reliance on polluting manufacturing processes. | Reduce cost by proposing a mass production system for hagfish filaments. | Reach out to the textile industry to find out about the biggest obstacles. Creating novel future textile materials with amazing mechanical properties & vivid colours |

![](https://static.igem.org/mediawiki/2020/c/ce/T--HKUST--sustainable_textile_system.png)

*Our sustainable Textile System Goals , (1.) Environmental: we hope to solve microfibres & toxic dye problems by replacing with biodegradable and non-toxic raw Materials for fibres & Dye, (2.) Economic: Our fiber production system lay foundational work on large scale productions of hagfish slime IF, and thus reducing cost. (3.) Social: By maintaining sustainability in environmental and economic development, consumers can enjoy high quality novel textile materials with incredible mechanical properties and vivid colours.*

# Project Design Overview

Previous efforts in synthesising recombinant hagfish fibres have yielded fibers of mediocre strength and quantity [7] [8]. We highlight three (3) major areas of improvement:

1. Fibre Yield

 2. Colour

 3. Fibre Quality

All of which are crucial if we are to produce a viable textile material for the clothings industry.

![](https://static.igem.org/mediawiki/2020/7/7b/T--HKUST--fibre_venn_diagram.png)

Our mass production system is comprised of five (5) aspects:

![](https://static.igem.org/mediawiki/2020/f/ff/T--HKUST--flowchart.png)

|  |  |
| --- | --- |
| **Aspects in Our System** | **Our Design** |
| 1. Fundamental research on Hagfish IF | WET LAB  Expression of Hagfish IF in vitro    DRY LAB  3D modelling of Basic structure of IF subunit  Dimer Model |
| 2. Expression | WET LAB  Expression methods : Inclusion Bodies VS Extracellular secretion  Comparing promoters : K880005 VS T7 Promoter |
| 3. Purification | WET LAB  T3SS Purification |
| 4. Dyeing | WET LAB  1. Type of Dye (Natural Dye & Chromoprotein) 2. Linkage of Dye (Covalently linking at different terminals & Non-covalent Binding Methods) 3. Colours Variety (Colour mixing) |
| 5. Fibre Formation | DRY LAB  Stress Model  Polymer formation model |

***1. Fundamental research on Hagfish IF***

In our fundamental research, we focused on the feasibility of synthesizing recombinant hagfish IF and developing the basis for structural modelling of IF proteins.

E. coli is our chassis for expressing the gene sequences of the 2 subunits, alpha and gamma of hagfish slime IF. According to previous teams’ (2014 UCC Ireland) results, it is expected that we will be able to produce synthetic hagfish IF visible under an optical microscope.

We predicted the structures of the 2 subunits of hagfish IF via homology modeling and ab initio modelling. Molecular dynamics simulations are then used to predict the hagfish IF dimer’s 3D protein structure.

***2. Expression***

We employ several different methods to increase the yield of hagfish IF proteins. It is hoped the addition of a signal peptide for extracellular secretion of target proteins will increase the yield of hagfish IF compared to the traditional method of protein extraction, which involves formation of inclusion bodies followed by cell-disruption. In addition, 2 different high expression strong promoter and RBS sets would be tested to compare the expression rates.

***3. Purification***

T3SS purification system is employed for efficient purification of hagfish IF subunits, subunits are ejected from the cell to the collection chamber. The usage of T3ss purification system is a cheaper approach to his-tag purification.

***4. Dyeing***

Various methods for dyeing hagfish IF with chromoproteins are explored. We attempt to link chromoproteins at either the N-termini or C-termini of each of our alpha and gamma hagfish IF subunits to pre-dye the hagfish IF threads. Alternative dyeing methods will be researched for a later stage.

We employ the subtractive colour mixing with three primary colours of chromoproteins (red, blue, and yellow) to expand the colour variations of IF fibres.

***5. Fibre Formation***

Molecular dynamics simulation is used for predicting the dimer formation of two Hagfish IF subunits with different modifications, such as linking chromoproteins with IF at the N-termini.

In addition, stress models which simulate IF fibres structural changes under stress are performed. This can help visualize the alpha to beta transition of IF during draw-processing, a process that can strengthen the mechanical properties of IF.

To further stimulate the structure of hagfish IF in reality, a polymer formation model of IF will be created in phase 2.

# Timeline

A summary of actions to be taken during different phases of our project is provided below:

1. In phase one, we focus on fundamental research: synthesizing the intermediate filament, providing modelling basis for dry lab, and brainstorming.

2. In phase two, we focus on exploring different methods for dyeing and purification of hagfish IF, as well as developing hardware and experimental improvements. More experimental and modeling results will be presented.

Unfortunately, we cannot conduct laboratory work due to Covid-19, thus we don't have enough time before the wiki freeze for synthesizing hagfish IF protein subunits. It is hoped that the situation would improve this winter (2020) that we might be able to do experiments to prove our concepts.

# Detailed Project Design

***1. Fundamental research (Focus on circuit)***

![](https://static.igem.org/mediawiki/2020/1/1a/T--HKUST--Parts_Table_1_1.png)

![](https://static.igem.org/mediawiki/2020/6/6d/T--HKUST--circuit_1.png)

*Figure (1) This construct is designed for expressing and producing our alpha and gamma subunits using the T7 promoter and terminator set. T7 is used because we want to try and replicate previous experiment results.*

For fundamental research, we first focus on synthesizing recombinant hagfish IF in vitro.

Our target is to replicate the previous 2014 team's (UCC Ireland) results in the lab.

Hagfish (Eptatretus stoutii) Thread biopolymer filament, including alpha (BBa\_K3581000) and gamma (BBa\_K3581001), is already codon optimized for e coli expression by the 2014 UCC team.

We first clone the 2 subunits from 2014 teams into the high copy pSB1-C3 vector separately. Strong promoter (T7 promoter BBa\_I712074) and strong RBS combination (T7 RBS BBa\_B0030) is used to ensure high expression level, terminated by T7 terminator (BBa\_B0016).

The reason for choosing T7 parts for our expression is to replicate results of a previous experimental data (2014 UCC) which also used T7 parts by secreting gamma subunits of hagfish intermediate filaments.

6x His tag which is inserted in the C terminus of protein to allow purification and identification.A TEV protease cleavage site (amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)) is placed upstream of the 6xHis-tag to facilitate tag removal.

Each plasmid is being transformed into separate e. coli culture (Strain : DH5Alpha).
For more details about experimental protocols , please visit [Wet Lab](https://2020.igem.org/Team%3AHKUST/Experiments).

![](https://static.igem.org/mediawiki/2020/1/1d/T--HKUST--Mr_e_coli_animated.gif)

Here is our Dearest Mr. E Coli, he shows us how is our IF is produced

We predicted the structures of the 2 subunits of hagfish IF via ab initio modelling, molecular dynamics simulations are then used to predict the hagfish IF dimer’s 3D protein structure.

For more details about modelling, please visit [Dry Lab](https://2020.igem.org/Team%3AHKUST/Model).

***2. Expression***

To increase protein yield, 2 strong constitutive promoters with RBS, BBa\_K880005 and BBa\_I712074, are incorporated in the construct. Comparison of the protein yield of the 2 promoters can help us determine which construct is more suitable to maximise protein yield.

The usual method of collection of hagfish intermediate filament (IF) is as follows: subunits are first expressed in the cytoplasm inside E. coli. The insoluble form of subunits will aggregate and lead to the formation of inclusion bodies inside the cell. Hagfish IF subunits are then retrieved by cell lysis, where the bacterial cell is disrupted and all cellular contents inside are released; upon further purification, pure hagfish IF subunits could be collected.

However, results from previous research using this approach shows that protein expression yield for hagfish IF is low, in the range of 40-50 mg per liter culture [9]. This yield is far from adequate for producing a viable fabric. Inclusion body aggregation might increase the metabolic load, which could decrease protein expression efficiency [10]. Not to mention the whole process is labor intensive.

Therefore we propose an alternative approach to boost the efficiency of hagfish IF expression and collection. We incorporated a signal peptide, either HlyA (BBa\_K3581003) or TorA (BBa\_K3114005) into our circuit downstream of our IF subunits at the C-terminus. These 2 signal peptides are chosen for comparison because each has its own advantages and limitations.

(1) Signal peptide HlyA (BBa\_K3581003) facilitates protein secretion via the type 1 secretion pathway, allowing our hagfish subunits to be transported from cytoplasm to extracellular space directly. However, HlyA is not cleaved by the bacterial cell when it is secreted out. It has to be cleaved out along before purification by Ni-NTA chromatography so that the his-tag would interact properly with the resin.

(2) Another signal peptide TorA (BBa\_K3114005) facilitates protein secretion via the TorA secretion system into the periplasm. Osmotic shock can then be applied to release the periplasmic proteins. In contrast with HlyA, TorA will be cleaved by the cell during secretion. The periplasmic proteins can be used for Ni-NTA chromatography directly.

The advantage of incorporating a signal peptide is that e. coli are not killed but are instead retained for continuous production of fiber proteins while also providing a simple collection process. We hope that by comparing different protein expressions and collection methods, a more practical approach could be determined.

The signal peptides are flanked with HindIII cut sites. This allows us to convert the mode of collection from protein secretion back to the conventional approach by digestion followed by ligation. Therefore we could use the same construct for different experiments.

![](https://static.igem.org/mediawiki/2020/4/4b/T--HKUST--Pdesign3.png)
![](https://static.igem.org/mediawiki/2020/a/a2/T--HKUST--Pdesign4.png)

*Figure (2). Inclusion body approach requires cell lysis which kills the cell. However, using a signal peptide, only our IFs are secreted out of the cell*

![](https://static.igem.org/mediawiki/2020/1/1a/T--HKUST--Parts_Table_1_1.png)

![](https://static.igem.org/mediawiki/2020/6/6d/T--HKUST--circuit_1.png)

*Figure (3) IF expression without signal peptide , inclusion body. Here we will use T7 promoter set for both alpha and gamma subunits*

![](https://static.igem.org/mediawiki/2020/c/c5/T--HKUST--composite_parts_4.png)

![](https://static.igem.org/mediawiki/2020/5/57/T--HKUST--composite_parts_3.png)

*Figure (4) IF expression with signal peptide, extracellular secretion. Here we will use T7 promoter set for both alpha and gamma subunits*

Comparing promoters : K880005 VS T7 Promoter

![](https://static.igem.org/mediawiki/2020/1/1a/T--HKUST--Parts_Table_1_1.png)

![](https://static.igem.org/mediawiki/2020/6/6d/T--HKUST--circuit_1.png)

*Figure (5) IF expression comparing promoters, T7 promoter. Here alpha and gamma subunits are expressed using the T7 promoter set by inclusion body expression method.*

![](https://static.igem.org/mediawiki/2020/b/b8/T--HKUST--Parts_Table_2_2.png)

![](https://static.igem.org/mediawiki/2020/8/80/T--HKUST--Parts_Table_2_1.png)

*Figure (6) IF expression comparing promoters, K880005. Here alpha and gamma subunits are expressed using the K880005 promoter set by inclusion body expression method.*

***3. Purification***

Our hagfish intermediate filaments (IF) would be of no use to the textiles industry if there is no way to efficiently mass produce it. In previous sections we have described protein expression methods that could produce IF proteins continuously. Here we propose a purification method that could save us time and money. Our proposed implementation is largely inspired by the purification system developed by Team: UCopenhagen 2018, which has its theoretical basis in the bacterial type III secretion system (T3SS).

Overview of the T3SS purification system

Type III secretion system (T3SS) are utilized by many gram-negative pathogens (which includes e. coli) to detect eukaryotic organisms and secrete proteins into them. In T3SS, a structure termed injectisome is a double-membrane-spanning syringe-like structure, which roughly divides into three parts: an extracellular needle-like appendage, associated cytoplasmic and membrane-bound secretion machinery. All three parts work collectively to deliver effector proteins into the cytosol of the eukaryotic host cell. Usually, the protein is secreted in an unfolded conformation, and the secretion requires both ATP and proton-motive force. It also involves an N-terminal secretion signal to guide the effector protein through the injectisome.

T3SS-based purification system in our project

To implement the T3SS purification system into our project, two major steps are involved:

1. Insertion of a T3SS signal sequence in our genetic construct:

We will insert a T3SS signal sequence immediately after the ribosome-binding site (RBS) of our circuit, so that the IF protein expressed would be attached to a T3SS signal peptide. The signal peptide would then guide our IF to reach the injectisome.

2. Transforming Synthetic Injector E. coli (SIEC) with IF subunit gene:

The T3SS genes will be incorporated into the bacterial genome, forming the SIEC. And the subunits gene will be carried by the plasmid vectors. The transformed cells will adsorbed on an artificial membrane via the action of the injectisome. The injectisome would then penetrate the artificial membrane. This allows our IF subunits, tagged with the T3SS signal peptide, to cross the artificial membrane and reaching the collection chamber.

![](https://static.igem.org/mediawiki/2020/b/bb/T--HKUST--tsss_hardware.png)

*Figure (7): Schematic representation of the T3SS purification hardware.*

## ***4. Dyeing***

After expression and purification of our hagfish recombinant IF proteins, pre-dyeing of the proteins will be the next step in our project.

Our target is to successfully produce a myriad of colored fibres while maintaining the incredible tensile mechanical properties in hagfish IF. This will bring our products closer to a viable material for clothing, while at the same time reducing waste water and toxic chemical pollution generated from traditional dyeing processes.

Here we describe our dyeing system in 3 main areas

1. Types of Dye
2. Linkage of Dye
3. Colours Variety

Each of these areas will be explored in detail in phase 2.

### 1. Types of Dye (Chromoproteins & Natural Dye)

Several criteria were considered when we decided which dyes to test:

Criteria of dye

* -Is it harmful or toxic?
* -Is it biodegradable?
* -Does it fade over time?

|  |  |
| --- | --- |
| **Criteria** | **Descriptions/problem to solve/application in our project/** |
| Ability to biodegrade | A biodegradable dye would be the perfect match for a biodegradable fibre. |
| Toxicity | A toxic or harmful dye would not be suitable for everyday wear. |
| Resistance to fasting (fading due to oxidation) | A dye that loses its colors quickly would not be suitable for clothes, which are supposed to last long. We realise this criteria is in conflict with row 1 (ability to biodegrade), but we are trying to strike a balance. |

We chose chromoprotein to be our preferred dye IF fibres. One reason is that chromoproteins are encoded by a single gene only, which allows protein expression to be relatively easy compared to engineering the entire complex metabolic pathway of organic dyes. This simpler dyeing method introduces less variables to the experiments. Also, unlike fluorescent proteins, chromoproteins can produce vivid colours without a UV-light source. This also makes chromoprotein more suitable for application in dyeing clothes.

A total of 9 chromoproteins were selected and ordered from Twist Bio for our dyeing experiments. The basic information of these chromoproteins are summarized in the following table:

|  |  |  |
| --- | --- | --- |
| **Primary Colours** | | |
| Red Chromoprotein | Blue Chromoprotein | Yellow Chromoprotein |
| ![](https://static.igem.org/mediawiki/2020/4/48/T--HKUST--Pdestab1.png) | ![](https://static.igem.org/mediawiki/2020/2/2b/T--HKUST--chromo2.png) | ![](https://static.igem.org/mediawiki/2020/b/b6/T--HKUST--chromo3.png) |
| BBa\_K592012 eforRed | BBa\_K864401 aeBlue | BBa\_K592010 amilGFP |
| BBa\_E1010 mRFP1 | BBa\_K592009 amilCP | BBa\_K1943001 fwYellow |
| **Secondary Colours** | | |
| Purple Chromoprotein | Green Chromoprotein | Orange Chromoprotein |
| BBa\_K mRFP violet mutant | BBa\_K592011 Cj Blue | BBa\_K1033913 scOrange |

After consulting Dr. Fudge, we realized that using chromoproteins for dyeing could be problematic, as it may affect polymer formation when linked at the termini of the subunits. He suggested the use of dye with smaller molecular size : congo red, but after consideration, we decided not to use it because of its carcinogenic properties. Instead, we would use an alternative natural dye such as indigo. Natural dyes are chemical dyes with a smaller molecular size. It is hoped that it can bind to our IF without affecting polymerization of hagfish IF. We will be exploring more about this idea in phase 2.

We also reconsidered our choice of chromoproteins, placing stronger emphasis on smaller size and small oligomeric states because we would still try chromoproteins while minimizing the effect on IF polymerization.

### 2. Linkage of Dye (Covalently links to terminals & non-covalently binding)

To bind our chromoprotein to our IF, we first tried to covalently link the chromoproteins at different terminals in each subunit:

(1) Linking at N-terminal for both subunits (Alpha & Gamma)

(2) Linking at C-terminal for both subunits (Alpha & Gamma)

(3) Linking at N-terminal for Alpha subunits and Linking at C-terminal for Gamma subunits

(4) Linking at N-terminal for Gamma subunits and Linking at C-terminal for Alpha subunits

The thinking behind trying different combinations is to explore the effect of linking chromoprotein at different positions on fibres formations. This characterization could inform later teams, should they attempt to make fusion protein with hagfish IF subunits.

![](https://static.igem.org/mediawiki/2020/8/82/T--HKUST--circuit_8.png)

*Figure (8). 2 cut sites are included in our genetic circuit: Acc65I is added at the N-terminal (between RBS and subunits) while AscI is added at the C-terminal (between subunits and his-tag). Both sites allow the insertion of chromoproteins, which are ordered in a separate fragment flanked by the same cut sites.*

|  |  |  |
| --- | --- | --- |
| **Both subunits linking at N terminus** | **Both subunits linking at C terminus** | **Each subunit linking at different terminal** |
| ![](https://static.igem.org/mediawiki/2020/c/c0/T--HKUST--Pdes1.png) | ![](https://static.igem.org/mediawiki/2020/3/35/T--HKUST--Design_Table_2.png) | ![](https://static.igem.org/mediawiki/2020/c/c0/T--HKUST--Pdes1.png) ![](https://static.igem.org/mediawiki/2020/3/35/T--HKUST--Design_Table_2.png) |
| ![](https://static.igem.org/mediawiki/2020/7/78/T--HKUST--Design_Table_4.png) | ![](https://static.igem.org/mediawiki/2020/d/d5/T--HKUST--Design_Table_5.png) | ![](https://static.igem.org/mediawiki/2020/1/14/T--HKUST--Design_Table_6.png) |

However, a big concern for linking chromoprotein to IF covalently through peptide bonds is that this linkage may hinder the formation and self assembly of IF subunits into a polymer, and therefore adversely affecting the mechanical properties of our fibre.

Therefore, other methods for linking chromoprotein non-covalently will be explored in phase 2. By comparing various methods, we aim to optimize our dyeing system to produce high quality coloured fibres.

In the dry lab part, one 3D protein structure example of the fusion protein is being generated with the help of CHIMERA software. Both subunits (Alpha & Gamma) are linked with Blue chromoprotein aeBlue at N-terminals by the peptide bond.

In phase 2, we will be applying this model in VMD simulation to estimate the stability of chromoproteins linkage to our IF in different conditions, for example, testing its heat stability when heating to certain degree, which can provide us information on the optimal temperature of using the coloured IF and its tendency of colour fading when in high temperature.

![](https://static.igem.org/mediawiki/2020/8/82/T--HKUST--structure_of_fusion_protein.png)

Figure (9) 3D protein structure of fusion protein is generated by CHIMERA, Here Each IF subunit is connected with Blue Chromoproteins (aeBlue) at N-terminal. The dimeric structure is shown

### 3. Colours Variety (Using Distinct Colours & Colour Mixing)

After chromoproteins are integrated into the circuit to grant the threads vivid colours, Experiments to assemble subunits with different coloured chromoprotein will be performed to explore the possibility of colour mixing, thereby expanding our range of possible colours.

Our basic colour mixing system is to link different colours of chromoproteins in each subunit. Each subunit (Alpha & Gamma) is connected with chromoproteins at N-terminal and forms dimer, dimer will form tetramers and finally bound together as an entire intermediate filaments polymers. Thus by mixing in different ratios, we hope to create fibres with a variety of colours.

![](https://static.igem.org/mediawiki/2020/4/4c/T--HKUST--Color_mixing_1.png)

Figure (10), Colour mixing by linking different colour of chromoproteins on each subunits (for example, Red chromoprotein on Alpha subunit, Blue chromoprotein on Gamma subunits) , Here chromoproteins are linked at N-terminal.

![](https://static.igem.org/mediawiki/2020/6/61/T--HKUST--Design_Figure_11.png)

Figure (11). This figure shows how mixing of colours produces different colours of Intermediate filaments theoretically. Here Blue chromoproteins is linked on gamma subunits, Red chromoproteins is linked on alpha subunits, the entire intermediate filaments is hoped to be purple in colour. (CP= Chromoproteins)

![](https://static.igem.org/mediawiki/2020/8/86/T--HKUST--Design_Figure_12.png)

Figure (12). 3 primary colours (Red, Blue, Yellow) are being used to link to each subunit. By linking different colour of chromoprotein to each subunits and mixing colour with 1:1 ratio, it is hope to generate 3 secondary colour (Green, Purple, Orange)

More theory supporting our colour mixing system will be illustrate in [DRY LAB](https://2020.igem.org/Team%3AHKUST/Model) Page

## ***5. Fibers Formations***

Normally, our IF fibres should undergo a tensile test in the laboratory with real equipment to determine their tensile properties and accordingly propose the possible applications.

![](https://static.igem.org/mediawiki/2020/f/f4/T--HKUST--Design_Figure_13.png)

Figure (13): A tensile testing equipment in the laboratory which could measure mechanical properties and provide strain for draw-processing

Under the COVID-19 pandemic, however, we could hardly produce the IF fibre themselves, not to mention carrying out an actual test on them. Hence, we turnt our focus on in silico [modeling](https://2020.igem.org/Team%3AHKUST/Model). We managed to predict the three-dimensional structure of hagfish slime intermediate filament dimer protein, and under the assumption that the dimer protein is the functional subunit of IF fibres, we tried to reveal the real-time conformational changes during draw-processing at the atomic level and also estimate the tensile properties of our product. In phase 2, we will try to construct the IF bundle polymer and demonstrate more accurate and detailed molecular basis of the outstanding tensile properties IF fibres possess.

## References

[1] J. Lim, D S. Fudge, et al. “Hagfish slime ecomechanics: testing the gill-clogging hypothesis” The Journal of Experimental Biology 209, 702-710, Published by The Company of Biologists 2006

[2] D S Fudge, S Hillis, N Levy and J M Gosline. “Hagfish slime threads as biomimetic model for high performance protein fibres” Bioinsp. Biomim. 5 (2010) 035002 (8pp),IOP PUBLISHING

[3] T.Vehoff, A.Glišović, H.Schollmeyer, A.Zippelius, T.Salditt. “Mechanical Properties of Spider Dragline Silk: Humidity, Hysteresis, and Relaxation. Biophysical Journal Volume 96, Issue12, 15 December 2007, Pages 4425-4432

[4] L Cheng, X Tong, et al. “Natural Silkworm Cocoon Composites with High Strength and Stiffness Constructed in Confined Cocooning Space” Polymers (Basel) 2018 Nov; 10(11): 1214

[5] J. Pérez‐Rigueiro, C. Viney, J. Llorca, M. Elices. “Silkworm silk as an engineering material” Journal of Applied Polymer Science/ Volume 70, Issue 12

[6] V K Gupta. “Fundamentals of Natural Dyes and Its Application on Textile Substrates” Chemistry and Technology of Natural and Synthetic Dyes and Pigments,2019

[7] UCC Ireland. 2014. Sea DNA. iGEM wiki. https://2014.igem.org/Team:UCC\_Ireland/Projects\_SeaDNA.html

[8] F. Jing, G. A. Paul; P. Andrea, H. Nils, Lim, C. Teck; H. J. Matthew, M. Ali. 2017. Artificial hagfish protein fibers with ultra-high and tunable stiffness. doi:10.1039/c7nr02527k

[9] Fu, J. (2016). Biomimetic engineering of materials based on hagfish slime thread proteins. Doctoral thesis, Nanyang Technological University, Singapore.

[10] Jim Klein and Prasad Dhurjati. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995. Protein Aggregation Kinetics in an Escherichia coli Strain Overexpressing a Salmonella typhimurium CheY Mutant Gene. 61(4).

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:HKUST/Experiments

iGEM HKUST

[ ]

[![](https://static.igem.org/mediawiki/2020/c/c5/T--HKUST--logo.png)](https://2020.igem.org/Team%3AHKUST)

* Project

  + [Description](https://2020.igem.org/Team%3AHKUST/Description)
  + [Design](https://2020.igem.org/Team%3AHKUST/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AHKUST/Contribution)
  + [Improvements](https://2020.igem.org/Team%3AHKUST/Improvements)
  + [Safety](https://2020.igem.org/Team%3AHKUST/Safety)
* [Dry Lab](https://2020.igem.org/Team%3AHKUST/Model)

  + [Protein Structure Pretiction](https://2020.igem.org/Team%3AHKUST/Model#heading-2)
  + [Molecular Dynamics Simulation](https://2020.igem.org/Team%3AHKUST/Model#heading-3)
  + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Model#heading-4)

  * [Wet Lab](https://2020.igem.org/Team%3AHKUST/Experiments)

    + [Description](https://2020.igem.org/Team%3AHKUST/Experiments#heading-1)
    + [Cloning](https://2020.igem.org/Team%3AHKUST/Experiments#heading-2)
    + [Expression & Purification](https://2020.igem.org/Team%3AHKUST/Experiments#heading-3)
    + [Thread Formation](https://2020.igem.org/Team%3AHKUST/Experiments#heading-4)
    + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-5)
    + [Draw-Processing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-6)
  * Human Practice

    + [Implementation](https://2020.igem.org/Team%3AHKUST/Implementation)
    + [Integrated Human Practice](https://2020.igem.org/Team%3AHKUST/Human_Practices)
    + [Collaborations](https://2020.igem.org/Team%3AHKUST/Collaborations)
    + [Education](https://2020.igem.org/Team%3AHKUST/Education)
    + [Partnership](https://2020.igem.org/Team%3AHKUST/Partnership)
    + [Entrepreneurship](https://2020.igem.org/Team%3AHKUST/Entrepreneurship)
  * Team

    + [Team Members](https://2020.igem.org/Team%3AHKUST/Team)
    + [Attributions](https://2020.igem.org/Team%3AHKUST/Attributions)
  * Parts

    + [Parts Overview](https://2020.igem.org/Team%3AHKUST/Parts)
    - [Basic Parts](https://2020.igem.org/Team%3AHKUST/Basic_Parts)
    - [Composite Parts](https://2020.igem.org/Team%3AHKUST/Composite_Parts)
  * Awards

    + [Award Criteria](https://2020.igem.org/Team%3AHKUST/Award)
    + [Achievements](https://2020.igem.org/Team%3AHKUST/Achievements)

# Wet Lab

* [## Basic Lab Plans](#heading-1)
* [## Cloning](#heading-2)
* [## Expression and Purification](#heading-3)
* [## IF assembly and thread formation](#heading-4)
* [## Colour Mixing](#heading-5)
* [## Draw-processing and mechanical enhancement](#heading-6)

# Basic Lab Plans

Sadly, due to the outbreak of COVID-19, access to the laboratory was restricted. Still, we made sure to plan out our lab work in detail. The following is a brief overview of our plans.

In this phase, our lab works were centered at:

1. The overexpression of recombinant hagfish slime intermediate filament(I) subunits
2. Fusion of chromoprotein on IF subunits for color mixing
3. Draw-processing for properties enhancement and measurement of thread mechanics

![](https://static.igem.org/mediawiki/2020/5/50/T--HKUST--experiments_flowchart.png)

Fig 1. Basic Lab Plans

# Cloning

All clones utilized the high copy-number pSB1C3 backbone as transformation vectors to carry the two hagfish slime intermediate filament (IF) subunits, 6x his-tag, secretion peptide, and chromoprotein genes. Most DNA fragments are synthesized by IDT while some chromoprotein sequences were ordered from Twist Bioscience. For detailed information on the BioBrick we used and all clones we constructed as mentioned follows, click [Parts](https://2020.igem.org/Team%3AHKUST/Parts) to see.

All fragments are geared with a standard prefix, with EcoRI, Xbal restriction site, and a standard suffix, with SpeI, PstI restriction site. Chromoproteins genes are additionally flanked by Acc65I and AscI restriction sites for its insertion upstream or downstream of the IF subunits sequence respectively.

The first objective, boosting expression efficiency of the subunits, is done by testing different strong promoter sets, and by inserting a secretion peptide after our gene of interest. There are two promoter-RBS sets, K88 (BBa\_K880005) and T7 (BBa\_I712074). We hope to identify and use the one with higher translational efficiency for subsequent experiments. Another approach is, by tagging the subunits with a short secretion peptide, TorA (BBa\_K3114005) or HlyA (BBa\_K554002), we hope to enhance protein expression yield by mitigating inclusion body formation. (Sørensen and Mortensen, 2005).

Fusion of chromoprotein for coloration of the thread is another focus of our project. After identifying the most efficient construct, chromoproteins of different CMY colors (blue/red/yellow) are being linked to the subunits through the Acc65I restriction site at the N-terminal, or AscI restriction site at the C-terminal.

Each recombinant plasmid is then transformed into E. coli. (DH5α strain). The cells were made competent with the inoue method. The transformed colonies are cultivated, and colony PCR is performed. Successfully recombinant plasmids are purified with the Thermo Scientific GeneJET Plasmid Miniprep Kit for subsequent use.

# Expression and Purification

Same E. coli. Strand (DH5α) as mentioned in the gene cloning part is used for protein expression. After transforming the competent cells with the desired recombinant plasmids, pre-culture containing: LB, chloramphenicol and a picked single colony is incubated overnight at 37 °C with vigorous shaking.

2ml of the pre-culture is then transferred into a larger container with fresh chloramphenicol-containing LB medium, and incubated under the same condition mentioned above, until OD600 reached 0.6-0.8. IPTG is then added to stimulate protein expression. The culture is further incubated for 3 hours.

For construct with no secretion peptide, the subunits produced are likely to aggregate in cells and form insoluble inclusion bodies. The purification process has to be done under denaturing conditions. Cells are harvested by centrifugation and lysed by vortexing in buffering solution with 8M urea as a denaturing agent. The clear supernatant is saved after centrifugation for further purification.

For construct with TorA secretion peptide inserted, the subunits will be expressed to the periplasm. Cells are harvested by centrifugation and resuspended in ice-cold magnesium sulphate. It serves as an osmotic shock agent which leads to the release of periplasmic proteins. The solution is then centrifuged and the supernatant is retained.

For construct with HlyA signal peptide, the tagged subunits will be expressed to the extracellular space. The supernatant will be used for purification after centrifugation.

Protein sizes and abundance are evaluated by SDS-PAGE before performing Ni-NTA column chromatography for the 6x His-tagged subunits. The yield of subunits is further analyzed using NanoDrop Microvolume Spectrophotometers and compared with different constructs.

![](https://static.igem.org/mediawiki/2020/2/28/T--HKUST--approaches_of_the_secretion_pathway_of_IF_subunits.png)

Fig 2. 3 approaches of the secretion pathway of IF subunits

The purified proteins are then cleaved with TEV protease to remove to 6x His-tag residues. Pure IF subunits can be obtained after running a size exclusion chromatography to exclude the his-tag residue and TEV protease in the solution. The resulting pure protein

As you might notice, the current purification method is tedious and too expensive to be applied in an industrial setting for mass production. Therefore, a novel purification method that does not rely on ordinary laboratory chromatography techniques is proposed. For more, please see project [design](https://2020.igem.org/Team%3AHKUST/Engineering) and future plans.

# IF Assembly and thread formation

The two purified subunits (alpha and gamma) are then mixed in a 1:1(w/w) ratio to undergo stepwise urea dialysis from 8 M to 0 M in Tris buffer of pH 8.4 at 4°C for self assembly of coil-coiled structure [1]. The resulting mixture can be freeze-dried and stored at 4°C for later use.

The freeze-dried mixture is then re-dissolved in 98% formic acid at 10% (w/v) concentration. Diluted with fresh formic acid, protein dope solutions prepared at different concentrations. Appropriate concentration of proteins dope solution is critical for the efficient production of mechanically well-performed threads. Increasing concentration of the solution manifests a positive relationship with thread diameter but is negatively correlated with threads of mechanical properties [2]. Dope solution concentration ranging from 5% to 10% will be used. Suitable amounts of the dope solution should be prepared upon demand to prevent degradation of IF in the doping solvent.

The simplest method to fabricate recombinant hagfish slime thread is to apply the protein dope solution on a surface of ice-cold magnesium chloride buffer and use a tweezer to slowly pick up the coagulated film from the solution interphase. The film will collapse due to gravity and forming a thread-like structure. The thread is then placed on a ventilated surface for overnight air dry.

More high-tech spinning methods, such as wet-spinning, electrospinning, and microfluidic devices can be investigated to enhance the efficiency of hagfish slime IF for mass production.

# Colour Mixing

In this phase, the colouration of threads is focused on the fusion of chromoproteins. As mentioned in the cloning section, chromoprotein is being inserted into the circuit after the most efficient construct is identified. The interference of thread properties by directly linking a chromoprotein is first being tested. After confirming the feasibility (i.e. the fusion protein can still form a proper thread), colour mixing by assembling subunits with different colours will begin. In order to identify the correct ratio of each colour and to better project the final colour of the resulting thread, the subtractive colour mixing model was worked in the [dry lab](https://2020.igem.org/Team%3AHKUST/Model).

In case if the linkage of chromoprotein greatly deteriorates the quality of the thread, or leads to self-assembly failure. Chromoproteins will be directly expressed and purified. By mixing different ratios of chromoprotein, solutions of different colours can be obtained. Hagfish slime thread is then directly immersed into the chromoprotein mixture for colouration. This method will be performed in parallel with dyeing the thread with natural dye, such as indigo. Other methods of non-covalently linking chromoproteins to IF will be explored in phase 2.

# Draw-processing and mechanical enhancement

It comes to the final step of the wet lab work, which is to artificially transform the alpha helix coiled-coil structure of hagfish slime intermediate filament to cross beta-sheet and eventually aligned beta-sheet. The molecular modelling of the transition is shown in the dry lab page The process is necessary for the thread to manifest outstanding mechanical properties that are comparable to spider silk.
Recombinant hagfish slime threads will be drawn from 25% to 100% with respect to its original length and their properties will be examined.

In case if the linkage of chromoprotein greatly deteriorates the quality of the thread, or leads to self-assembly failure. Chromoproteins will be directly expressed and purified. By mixing different ratios of chromoprotein, solutions of different colours can be obtained. Hagfish slime thread is then directly immersed into the chromoprotein mixture for colouration. This method will be performed in parallel with dyeing the thread with natural dye, such as indigo. Other methods of non-covalently linking chromoproteins to IF will be explored in phase 2.

![](https://static.igem.org/mediawiki/2020/6/6c/T--HKUST--tensile_testing.jpeg)

Fig 3. A tensile equipment in the laboratory which could measure mechanical properties and provide strain for draw-processing

# References

[1] F. Jing, “Biomimetic engineering of materials based on hagfish slime thread proteins,” 2016.

[2] A. Negishi, C. L. Armstrong, L. Kreplak, M. C. Rheinstadter, L.-T. Lim, T. E. Gillis, and D. S. Fudge, “The Production of Fibers and Films from Solubilized Hagfish Slime Thread Proteins,” Biomacromolecules, vol. 13, no. 11, pp. 3475–3482, 2012.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:HKUST/Results

iGEM HKUST

[ ]

[![](https://static.igem.org/mediawiki/2020/c/c5/T--HKUST--logo.png)](https://2020.igem.org/Team%3AHKUST)

* Project

  + [Description](https://2020.igem.org/Team%3AHKUST/Description)
  + [Design](https://2020.igem.org/Team%3AHKUST/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AHKUST/Contribution)
  + [Improvements](https://2020.igem.org/Team%3AHKUST/Improvements)
  + [Safety](https://2020.igem.org/Team%3AHKUST/Safety)
* [Dry Lab](https://2020.igem.org/Team%3AHKUST/Model)

  + [Protein Structure Pretiction](https://2020.igem.org/Team%3AHKUST/Model#heading-2)
  + [Molecular Dynamics Simulation](https://2020.igem.org/Team%3AHKUST/Model#heading-3)
  + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Model#heading-4)

  * [Wet Lab](https://2020.igem.org/Team%3AHKUST/Experiments)

    + [Description](https://2020.igem.org/Team%3AHKUST/Experiments#heading-1)
    + [Cloning](https://2020.igem.org/Team%3AHKUST/Experiments#heading-2)
    + [Expression & Purification](https://2020.igem.org/Team%3AHKUST/Experiments#heading-3)
    + [Thread Formation](https://2020.igem.org/Team%3AHKUST/Experiments#heading-4)
    + [Color Mixing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-5)
    + [Draw-Processing](https://2020.igem.org/Team%3AHKUST/Experiments#heading-6)
  * Human Practice

    + [Implementation](https://2020.igem.org/Team%3AHKUST/Implementation)
    + [Integrated Human Practice](https://2020.igem.org/Team%3AHKUST/Human_Practices)
    + [Collaborations](https://2020.igem.org/Team%3AHKUST/Collaborations)
    + [Education](https://2020.igem.org/Team%3AHKUST/Education)
    + [Partnership](https://2020.igem.org/Team%3AHKUST/Partnership)
    + [Entrepreneurship](https://2020.igem.org/Team%3AHKUST/Entrepreneurship)
  * Team

    + [Team Members](https://2020.igem.org/Team%3AHKUST/Team)
    + [Attributions](https://2020.igem.org/Team%3AHKUST/Attributions)
  * Parts

    + [Parts Overview](https://2020.igem.org/Team%3AHKUST/Parts)
    - [Basic Parts](https://2020.igem.org/Team%3AHKUST/Basic_Parts)
    - [Composite Parts](https://2020.igem.org/Team%3AHKUST/Composite_Parts)
  * Awards

    + [Award Criteria](https://2020.igem.org/Team%3AHKUST/Award)
    + [Achievements](https://2020.igem.org/Team%3AHKUST/Achievements)

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)