iGEM 2019 — Team Manchester

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Manchester/Notebook

UoM iGEM | Project Cutiful

![](https://static.igem.org/mediawiki/2019/f/f7/T--Manchester--Template-LoadingGif.gif)

[![navbar-logo](https://static.igem.org/mediawiki/2019/a/a2/T--Manchester--navbarlogo.png)](https://2019.igem.org/Team%3AManchester)

* PROJECT

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Description

  Brief Overview of Project](https://2019.igem.org/Team%3AManchester/Description)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Modelling

  Theory and Aims](https://2019.igem.org/Team%3AManchester/Model)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Timeline

  Chronological Project Layout](https://2019.igem.org/Team%3AManchester/Notebook)
* EXPERIMENT

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Colour

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Experiments)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Fragrance

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Fragrance)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Repair

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Repair)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Demonstration

  Our work in real life](https://2019.igem.org/Team%3AManchester/Demonstrate)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Safety

  In the Lab, and in Our Project](https://2019.igem.org/Team%3AManchester/Safety)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Measurement

  Special Prize](https://2019.igem.org/Team%3AManchester/Measurement)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Protocols

  Our protocols](https://2019.igem.org/Team%3AManchester/Protocols)
* PARTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Parts Collection

  Our New Biobricks](https://2019.igem.org/Team%3AManchester/Part_Collection)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Contribution

  Chracterization of Existing Biobricks](https://2019.igem.org/Team%3AManchester/Contribution)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Improvement

  Our Improved Biobricks](https://2019.igem.org/Team%3AManchester/Improve)
* HUMAN PRACTICES

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Integrated Human Practices

  Cutiful in the Wider world](https://2019.igem.org/Team%3AManchester/Human_Practices)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Public Engagement

  Protocols and results for colour](https://2019.igem.org/Team%3AManchester/Public_Engagement)
* TEAM

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Members

  Meet the Team](https://2019.igem.org/Team%3AManchester/Team)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Attributions

  Our Advisors, Contributors and Sponsors](https://2019.igem.org/Team%3AManchester/Attributions)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Collaborations

  Outreach Within iGEM Teams](https://2019.igem.org/Team%3AManchester/Collaborations)
* ACHIEVEMENTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Medal Criteria

  Where We Impress You](https://2019.igem.org/Team%3AManchester/Medal)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Judging Form

  At the Judges' Convenience](https://igem.org/2019_Judging_Form?team=Manchester)
* [![](https://static.igem.org/mediawiki/2019/archive/4/4c/20191005093133%21T--Manchester--tw-icon.png)](https://twitter.com/iGEM_Manchester)
  [![](https://static.igem.org/mediawiki/2019/archive/0/0f/20191005093741%21T--Manchester--ig-icon.png)](https://www.instagram.com/igem.manchester/)

![](https://static.igem.org/mediawiki/2019/thumb/3/3d/T--Manchester--Template-BackArrow.png/800px-T--Manchester--Template-BackArrow.png)

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That’s it, folks!

We hope you’ve enjoyed our journey through iGEM, full of sleep deprivation, last minute scrambling and a great deal of laughter. We’ve cried (more than once), we’ve gone home far too late (for a variety of reasons), and we’ve shared our food. The latter is, truly, the mark of our friendship.

From now onwards, we have to polish off our presentation, go out as a team once more before Boston (but only after we’ve caught up on all our sleep) and hope to see you all at the Jamboree. We’ll be found in Zone 3, Poster number: 263, and are presenting on Saturday in Room 304 at 3pm. Make a note: come and find us! Sara will be more than happy to give you cooking tips (her food, really, was delicious) and Jonathan will probably have a comment or two regarding how to keep fellow team-members from snatching your cookies. If you think you can dethrone Camilo’s place as #1 bowling champion, then please come along. We have ten days to manage it before Boston.

And after that?

Who knows. Let’s survive this first!

(Here be Manchester iGEM 2019.)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Manchester/Experiments

UoM iGEM | Project Cutiful

![](https://static.igem.org/mediawiki/2019/f/f7/T--Manchester--Template-LoadingGif.gif)

[![navbar-logo](https://static.igem.org/mediawiki/2019/a/a2/T--Manchester--navbarlogo.png)](https://2019.igem.org/Team%3AManchester)

* PROJECT

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Description

  Brief Overview of Project](https://2019.igem.org/Team%3AManchester/Description)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Modelling

  Theory and Aims](https://2019.igem.org/Team%3AManchester/Model)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Timeline

  Chronological Project Layout](https://2019.igem.org/Team%3AManchester/Notebook)
* EXPERIMENT

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Colour

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Experiments)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Fragrance

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Fragrance)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Repair

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Repair)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Demonstration

  Our work in real life](https://2019.igem.org/Team%3AManchester/Demonstrate)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Safety

  In the Lab, and in Our Project](https://2019.igem.org/Team%3AManchester/Safety)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Measurement

  Special Prize](https://2019.igem.org/Team%3AManchester/Measurement)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Protocols

  Our protocols](https://2019.igem.org/Team%3AManchester/Protocols)
* PARTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Parts Collection

  Our New Biobricks](https://2019.igem.org/Team%3AManchester/Part_Collection)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Contribution

  Chracterization of Existing Biobricks](https://2019.igem.org/Team%3AManchester/Contribution)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Improvement

  Our Improved Biobricks](https://2019.igem.org/Team%3AManchester/Improve)
* HUMAN PRACTICES

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Integrated Human Practices

  Cutiful in the Wider world](https://2019.igem.org/Team%3AManchester/Human_Practices)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Public Engagement

  Protocols and results for colour](https://2019.igem.org/Team%3AManchester/Public_Engagement)
* TEAM

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Members

  Meet the Team](https://2019.igem.org/Team%3AManchester/Team)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Attributions

  Our Advisors, Contributors and Sponsors](https://2019.igem.org/Team%3AManchester/Attributions)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Collaborations

  Outreach Within iGEM Teams](https://2019.igem.org/Team%3AManchester/Collaborations)
* ACHIEVEMENTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Medal Criteria

  Where We Impress You](https://2019.igem.org/Team%3AManchester/Medal)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Judging Form

  At the Judges' Convenience](https://igem.org/2019_Judging_Form?team=Manchester)
* [![](https://static.igem.org/mediawiki/2019/archive/4/4c/20191005093133%21T--Manchester--tw-icon.png)](https://twitter.com/iGEM_Manchester)
  [![](https://static.igem.org/mediawiki/2019/archive/0/0f/20191005093741%21T--Manchester--ig-icon.png)](https://www.instagram.com/igem.manchester/)

![](https://static.igem.org/mediawiki/2019/thumb/3/3d/T--Manchester--Template-BackArrow.png/800px-T--Manchester--Template-BackArrow.png)

* [Introduction](#intro)
* [Design](#colour)
* [Experimental and Results](#fragrance)
* [Conclusion](#kill)
* [Overall Conclusions](#allergen)
* [The Future](#skin)

# COLOUR

Operating within additive and subtractive colour theory, the ability of our chromophores to emit in the UV range and absorb in the visible spectrum makes Cutiful uniquely suited to be declined in a full range of hues, personnalisable and innovative. Manchester iGEM 2019 presents to you a hair dye able to blend in with natural tones during the day, lighting up the clubs by night!

Our Colour Module…

### In 7 bullet points:

### 1.

Seven new constructs - we achieved successful cloning of 7 newly designed constructs related to dye production in *E. coli*.

### 2.

Characterised Two Previous Parts - we have also re-used and provided further characterisation on 2 previously registered iGEM parts.

### 3.

New Tag For Hair Adhesion - we have adapted a hydrophobic tag into our constructs in order to provide wanted adhesive characteristics to the biosynthesised chromoprotein (dyes).

### 4.

New Composite sfGFP part - we have designed and characterised a new essential composite part, sfGFP with tet promoter.

### 5.

Characterisation of AmilCP - we verified the absorption maxima of AmilCP, a blue chromoprotein, as 588 nm.

### 6.

Characterisation of New Functional Part - we have characterised a new functional part (mRFP1 containing a C-terminal secretion signal (HylA) and the hydrophobic tag) proved through both quantitative (SDS-PAGE) and qualitative data.

### 7.

Bespoke Hue Palette - we achieved a wide palette of colours: visible both under normal white lighting and also under fluorescence (such as nightclub environment). ([Demonstration, Act III: Cutiful - into the wider world](https://2019.igem.org/Team%3AManchester/Demonstrate))

![](https://static.igem.org/mediawiki/2019/8/80/T--Manchester--Colours--4.png)

Figure 1. Hue Palette for Cutiful, generated by mixing mRFP1, amilCP and sfGFP in varying ratios. Altering visible light (left) and UV light (right).

#### Colours – the beginning

Or: How to learn spanish in three months. Also includes rainbows, late nights, a certain degree of madness, and enough experiments to make Benchling crash - though not necessarily in that order.

Hair dyes can cause damage to the end user as well as the environment. Products currently available on the market contain allergens, pollutants and potentially health-damaging compounds[(Project Description)](https://2019.igem.org/Team%3AManchester/Description). The use of a bacterial whole-cell system could provide a solution to these concerns. Our initial modelling to ensure colour coverage (based on molar extinction coefficients) determined the selection of our protein-based dyes[(Modelling, Act I: Colours)](https://2019.igem.org/Team%3AManchester/Model). Our genetically modified bacteria are able to secrete chromoproteins, which are designed to interact with the outer β-layer of the hair cuticle: becoming an alternative to dyes. After confirming attachment of DH5⍺ and BL21(DE3) to hair, we investigated how *E. coli* (transformed with our designed constructs) were able to produce chromoproteins containing a **novel hydrophobic tag**, able to interact with the cuticle[(Demonstration, Act II: Results)](https://2019.igem.org/Team%3AManchester/Demonstrate). This decreases the need for damaging agents, such as cuticle-opening bleach. Additionally, we also achieved a wide range of colours. Future experiments could easily provide a modular system able to produce a palette of colours as the consumer desires.

#### Prologue

“why do two colours, put one next to the other, sing?” - Pablo Picasso

We decided to initially produce 3 different colour proteins in order to create our bacterial-based hair dye. The selection of these proteins was based on our initial modelling on colour. Green sfGFP (which had been reported to look yellow under normal light, [BBa\_K1321337](http://parts.igem.org/Part%3ABBa_K1321337), ε=83 300 M-1cm-1) and blue AmilCP
([BBa\_K592009](http://parts.igem.org/Part%3ABBa_K592009), ε=87 600 M-1cm-1) were chosen for their high molar extinction coefficients, which promised to offer better coverage of the hair’s natural colour. In order to close the chromatic circle, a red colour protein seemed the obvious third choice, enabling us to generate varied hues through mixing (See page icon above for a schematic representation of the chromatic circle in  [additive colour theory](https://study.com/academy/lesson/additive-color-theory-definition.html)). Red mRFP1 ([Bba\_E1010](http://parts.igem.org/Part%3ABBa_E1010), ε=50 000 M-1cm-1) was chosen after further research, having already been successfully used by previous iGEM teams. Its extinction coefficient is slightly less than those of our first two chromophores, and indeed not the highest amongst red fluorescent proteins; DsRed, for example, has a higher extinction coefficient than mRFP1. However, mRFP1 matures over ten times faster than DsRed, and thus shows a similar brightness. Hence, it was chosen for its remarkable folding abilities. Models have predicted that, whilst AmilCP would be expected to create a more intense colour, sfGFP would produce the most noticeable effect[(Modelling, Act I: Colours)](https://2019.igem.org/Team%3AManchester/Experiments). Laboratory experiments have shown all three colours to express successfully, and colonies can be mixed to obtain a gradient, with mRFP1 offering the best coverage in the visible spectrum.

From each coloured protein we designed 3 different variants (as explained below).

We ordered the synthesis of 3 coloured gBlocks® from IDT, with the following general structure:

![](https://static.igem.org/mediawiki/2019/4/4e/T--Manchester--Colours--SBOL.png)

Figure 2. Schematic depiction of ordered gBlock® of DNA. Arrows represent the primers used for PCR amplification of each BioBrick. For each colour protein three variations were made: (purple) PCR of the stock BioBrick; (brown) Primers amplified the colour only and the forward binding primer also adds the RBS; (green) Colour protein preceded by both the RBS in this case situated in the ordered gBlock and a N-terminal secretion signal sequences (OmpA); (blue) Again, the RBS was added using primers and a C-terminal secretion signal (HylA) was added to this construct. Note that the promoter (tetR) and terminators (rrnB T1 terminator and T7 terminator were both located on the vector pBbB2c).

For each coloured protein we decided to produce 3 different constructs as schematically represented above. The colour alone construct (without tags) was used as a control for comparison as well as for further characterisation. For generation of the colour alone and C-terminal constructs, primers shown in brown and blue respectively were also required for the addition of the selected ribosome binding site ([BBa\_J34801](http://parts.igem.org/Part%3ABBa_J34801)) (see Figure 1). Hence, primers (see supplementary data) were used to amplify the inserts to make the required full constructs.

#### Act I: Design

“For colour is one of the most rapturous truths that can be revealed to man.” ― Harold Speed, Oil Painting Techniques and Materials

#### Scene 1: Secretion Signal Tags

We aimed at the production of both an N-terminal and a C-terminal secretion signal sequence, OmpA and HylA respectively, in order to maximise the chances of having a successful secretion system. Outer membrane peptide A (OmpA) is a conserved protein domain found at the outer membrane of many gram-negative bacteria such as *E. coli*. The OmpA secretion signal is located at the N-terminal region of the protein of interest and becomes trafficked through a type II secretion system. The signal peptide is cleaved by the secretion machinery in the plasma membrane of the bacterium. The resulting protein is secreted and released in an active mature form. This secretion signal peptide was therefore selected due to its reported high efficiency of secretion during high protein expression. The second chosen secretion signal was α-hemolysin (HylA) which has been previously characterised to provide efficient secretion of active correctly folded proteins to the culture supernatant in *E. coli*. This hemolysin secretion system is a well characterised type I secretion system which provides one-step translocation, from the bacterial cytoplasm to the extracellular medium without a periplasmic intermediate of tagged proteins. The secretion signal, unlike with OmpA, does not become cleaved after trafficking across the bacterial membrane.

Since we aimed at the creation of new hair dyes that would not damage the cortex of hair, we did not want our designed coloured protein-based dyes to infiltrate the cortex as this will lead to cuticle opening and weaken the hair itself. Therefore, both of our secreted variants also contained a novel hydrophobic tag adapted from Y3P2‐GFPuv consistent of YYYPP (where Y indicates tyrosine and P proline). This tag was originally used for chromatography during protein separation. However, we decided to adapt this to enable our coloured proteins to embed in the hydrophobic beta-layer of the cortex of hair.

#### Scene 2: Backbone of Choice

For coloured protein expression we selected the BglBrick plasmid **pBbB2c-GFP**. The plasmid uses a pBBR1 origin of replication, this is the second highest copy number plasmid with around 20 copies per cell from BglBrick plasmids library. We chose this backbone in order to maximise production and lower cell burden by cloning the hair repair (decapeptide) in future experiments. The promoter, located on the vector, is TetR ([BBa\_R0040](http://parts.igem.org/Part%3ABBa_R0040)), which regulates expression of our desired constructs. This is a strongly regulated promoter, minimises leakiness to avoid toxic protein aggregation and allows controlled expression. We additionally chose this vector as it does not possess any BsaI sites and hence it could be used for our chosen cloning, type IIS.

Therefore, from our 3 ordered DNA gBlocks® (IDT), we obtained, tested and characterised **7 new parts** and additionally, used **two previously characterised** iGEM parts for our experiments and for further characterisation. All these parts were cloned into pBbB2c and expressed through the tetR system in two selected *E. coli* strains (**DH5⍺** and **BL21(DE3)**).

#### Act II: Experimental and Results

“When the colours speak, close your mouth tight and open your eyes wide!” ― Mehmet Murat ildan

All nine colour constructs, sfGFP, mRFP1 and AmilCP with their respective signals (Alone, Colour with N-Terminal secretion signal, and Colour C-Terminal secretion signal), were treated identically during experimentation unless specified:

1.  [PCR](https://2019.igem.org/Team%3AManchester/Protocols) amplification of each desired construct.

![](https://static.igem.org/mediawiki/2019/2/21/T--Manchester--Colours--PCR.png)

Figure 3. Gel electrophoresis of all our colour parts amplified through PCR from ordered gBlocks ® for cloning. Non-specific bands removed through gel extraction when required (see supplementary data). Expected band sizes are provided on top of each band (in bp).

2. [Type IIS](https://2019.igem.org/Team%3AManchester/Protocols)(Golden Gate) assembly into the selected backbone (pBbB2c)

3.  [Transformation](https://2019.igem.org/Team%3AManchester/Protocols) into *E. coli* DH5⍺

4.  [Colony PCR](https://2019.igem.org/Team%3AManchester/Protocols) to test insertion and overnight inoculations of positive colonies

5. Plasmid purification  [(miniprep)](https://2019.igem.org/Team%3AManchester/Protocols) purification and  [restriction digestion](https://2019.igem.org/Team%3AManchester/Protocols) to ensure the positive colony PCR results do indeed contain the insert

6. [Sequencing](https://2019.igem.org/Team%3AManchester/Protocols) to verify cloning and ensure no mutations have been introduced

7. [Transformation](https://2019.igem.org/Team%3AManchester/Protocols) into BL21(DE3) cells.

8. Characterisation.

Quantitative data

Qualitative data

#### Scene 1: sfGFP

As described above, the selection of sfGFP was determined by its extinction coefficient which was investigated during our initial modelling. This meant that, as a result of its intensity of light absorption, it could provide a visible effect when coating hair: resembling a semi-permanent hair dye. Additionally, sfGFP had been already shown to auto-secrete which favoured its selection. However, as this diffusion could be lower than what we were looking for, we decided to also produce it following the same criteria as the other two proteins we chose, with both a N-terminal as well as a C-terminal secretion signal sequence (Figure 1).

![](https://static.igem.org/mediawiki/2019/a/aa/T--Manchester--Colours--3DGFP.png)

Figure 4. 3D  [structure of sfGFP](https://www.fpbase.org/protein/superfolder-gfp/)

Superfolder GFP, also known as sfGFP, is a GFP-derived green fluorescent protein. GFP is a protein isolated from the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. A series of mutations performed by Pédelacq et al. obtained a GFP variant able to **rapidly fold and mature**. This ultimately also lead to an enhanced fluorescence intensity.

Due to this improved properties we selected sfGFP (structure seen in Figure 3) as one of our hair dye colours. We therefore decided to build and characterise the 3 constructs depicted in Table 1.

Table 1. Constructed sfGFP parts. All parts provided are novel to the best of our knowledge. The colour alone construct ([BBa\_K2906000](http://parts.igem.org/Part%3ABBa_K2906000)) of sfGFP is under the regulation of the TetA/R promoter. This part was used not only for characterisation but also as a positive control. N-terminal ([BBa\_K2906001](http://parts.igem.org/Part%3ABBa_K2906001)) contains a hydrophobic tag designed for hair attachment as well as OmpA required for secretion; similarly C-terminal ([BBa\_K2906002](http://parts.igem.org/Part%3ABBa_K2906002)) follows the same structure, however in this case the signal HylA required for secretion.

| Name | Registry Code | BioBrick |
| --- | --- | --- |
| Colour alone | BBa\_K2906000 | ![](https://static.igem.org/mediawiki/2019/1/1a/T--Manchester--parts.registration--GFP1.png) |
| N-terminal | BBa\_K2906001 | ![](https://static.igem.org/mediawiki/2019/9/97/T--Manchester--parts.registration--GFP2.png) |
| C-terminal | BBa\_K2906002 | ![](https://static.igem.org/mediawiki/2019/6/62/T--Manchester--parts.registration--GFP3.png) |

Note. The C-terminal construct used had a deletion.

Multiple cloning attempts were not able to generate sfGFP C-terminal construct. Due to time constraints, experiments were performed with a mutated version of the sfGFP C-terminal construct. After sequencing four times, with different companies (two times per company), all results came back identical. Sequencing results are provided below.

![](https://static.igem.org/mediawiki/2019/0/04/T--Manchester--Colours--Seq.png)

Figure 5. sfGFP-HylA mutation in DNA sequence

The mutation obtained was an 8 bp deletion from position 31 to 39 bp of sfGFP, causing a frame-shift mutation. However, this deletion is found 49-57 bp away from the start codon, located immediately upstream of the hydrophobic tag.

We looked at the six different possible reading frames (file provided in our supplementary data), which allowed us to identify a possible 483 bp sequence which possesses a start codon 220 bp later than the sfGFP start codon, but is found to be in frame with the original unmutated sfGFP. This means a truncated form of the sfGFP protein could be expressed (as surprising results were obtained, see Figure 10). However, since the sfGFP chromophore (TYG where T is Thr; Y is Tyr and G denotes Gly) is not located on this reading frame it seems unlikely that this truncated protein contributes to our surprising plate reader results. Currently, we are unable to confirm a possible Open Reading Frame (ORF) for this potentially truncated yet functional green fluorescent protein.

Following the methods stated, we successfully achieved the cloning of our designed parts into pBbB2c and transformed them into *E. coli* DH5⍺ and BL21(DE3) respectively. Initially, from fresh transformant plates (LBA + Cm, incubated at 37ºC overnight) a single colony was picked and inoculated in 5 mL of LB media for overnight growth (37ºC with shaking at 180 rpm). As a negative control we used an *E. coli* TOP10 strain because it does not express colour. This strain was chloramphenicol resistant and was available in-house. The negative control followed the same protocol as sample cultures. After 16-18 hours growth, the cultures were re-inoculated as a 1:100 dilution in 50 mL LB containing 34 µg/ml chloramphenicol (500 mL conical flask). The cultures were grown at 37ºC at the same shaking conditions. The growth was monitored up to a desired OD600 of ~0.6. Once the bacterial samples had reached the desired OD600, a 5 mL cell pellet was obtained (Fig. 6) and 5 mL sample for SDS. Samples were induced with 100 nM of anhydrotetracycline since expression was regulated by the tetR/tetA operator. 200 μL of each culture was transferred onto one well of a 4titude black, clear-bottom sterile 96-well plate, in triplicates. A plate reader (CLARIOstar®, BMG Labtech) was used to measure OD600 and RFU every 15 minutes overnight. The remainder of the culture in the 500 mL flasks were incubated at 30ºC with shaking. Every hour for 4 hours after induction, a 5 mL bacterial culture was obtained to form a pellet (see Figure 4). Lastly, after overnight incubation at 30ºC with shaking at 180 rpm, a 5 mL sample was obtained for **SDS-PAGE** and an additional 5 mL cell pellet was also obtained. **Microplate reader** results were then collected, analysed and plotted.

We achieved some level of expression of all three novel constructs. However, the secretion mechanism requires further investigation. We did achieve the expression and secretion of colour alone ([BBa\_K2906000](http://parts.igem.org/Part%3ABBa_K2906000)). The exact mechanism behind this secretion process also requires of further investigation, but, our analysis clearly shows the presence of this product in the supernatant.

#### QUANTITATIVE RESULTS:

Characterisation using microplate reader:

Once samples had been induced, including the negative control, 200 μL of each culture was transferred onto a 4titude black, clear-bottom sterile 96-well plate. Of each sample 3 technical replicates were performed. Then, the plate reader (CLARIOstar®, BMG Labtech) was used to measure OD600 and RFU every 15 minutes overnight (12-13 hrs). The following morning, the data was obtained, analysed and plotted by blank-correction using both LB-containing microplate wells as well as TOP10 cells for correction. TOP10 was used as a control since it does not express fluorescence.

Initially we looked at the standard growth curve obtained by the measurement of OD600.

![](https://static.igem.org/mediawiki/2019/7/7f/T--Manchester--Colours--GFPOD.png)

Figure 6. The plot shows the growth of bacteria over time determined by its OD600. Measurements were taken every 15 minutes following induction. The negative control, TOP10 was also induced.

This allowed us to visualise the normal bacterial growth of the untagged (colour alone) and C-terminal secretion signal transformed bacteria. However, there was an unexpected drop in optical density, and therefore in bacterial population for the N-terminal secretion signal bacteria.

We then looked at the **fluorescence measurements**, which were normalised by dividing them by their OD600. Below we show the processed results.

RFU/OD:

![](https://static.igem.org/mediawiki/2019/5/50/T--Manchester--Colours--GFPRFU.png)

Figure 7. The plot shows the mean RFU/OD from three replicates of each construct expressed in *E. coli* DH5⍺ and BL21(DE3). The OD was measured at 600 nm and GFP fluorescence was measured at Ex ƛ 485, Em ƛ 510, every 15 minutes for 13 hours. The RFU values were normalised by the OD600 and the triplicates averaged. All values have been blank-corrected. A total of 52 recordings were made per well, with three wells per construct.

From Figure 7 we can observe that both the colour alone as well as the N-terminal sfGFP constructs were able to produce fluorescence. The non-tagged construct (colour alone) had a much higher output. Additionally, the lower fluorescence levels could also be a result of the normalisation as a population decline was observed (Figure 3). On the other hand, the C-terminal construct did not show any apparent fluorescence above basal background levels in either of the tested E. coli strains.

SDS-PAGE:

Besides the characterisation of the absorbance and emittance spectra of all our sfGFP parts, we also wanted to visualise if the protein was being **over-expressed** and **secreted** across the bacterial membrane. For that reason, a 5 mL sample was obtained both at time 0 before induction, and following induction and overnight incubation at 30ºC.

We first calculated the expected sizes of sfGFP on all our desired constructs: We determined that the colour alone was expected to have a 26.8 kDa molecular weight, whereas the N-terminal tag and cleaved C-terminal tag of 29.2 kDa (or 27.7 kDa when secretion signal becomes fully cleaved) and 33.2 kDa respectively. Then samples were run on 10% gels following  [SDS protocol](https://2019.igem.org/Team%3AManchester/Protocols).

![](https://static.igem.org/mediawiki/2019/1/1c/T--Manchester--Colours--NOBGGFPSDS1.png)

![](https://static.igem.org/mediawiki/2019/8/82/T--Manchester--Colours--NOBGGFPSDS2.png)

![](https://static.igem.org/mediawiki/2019/b/b7/T--Manchester--Colours--NOBGGFPSDS3.png)

![](https://static.igem.org/mediawiki/2019/2/20/T--Manchester--Colours--NOBGGFPSDS4.png)

![](https://static.igem.org/mediawiki/2019/0/08/T--Manchester--Colours--NOBGGFPSDS5.png)

![](https://static.igem.org/mediawiki/2019/b/b5/T--Manchester--Colours--NOBGGFPSDS6.png)

Figure 8. SDS-PAGE gels (Mini-PROTEAN TGX Stain-Free Precast gels 10%). A 5 mL sample was taken from each construct before induction (negative control) and after overnight inoculation at 30ºC following induction with anhydrotetracycline (O/N). The expected molecular sizes: Colour alone (26.8 kDa); N-terminal (29.2 kDa or 27.7 when fully cleaved and secreted) and C-terminal of 33.2 kDa. All samples were checked for protein expression. Supernatant, soluble and insoluble fractions were all analysed during this experiment.

The gels in Figure 8 show a comparison between time 0, when samples were not induced and induced after overnight incubation at 30ºC with shaking at 180 rpms. In order to aid visualisation we provide a summary table below.

|  | | | | DH5ɑ | | | | | | BL21(DE3) | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | | Negative control (TOP10) | | sfGFP alone | | sfGFP N terminal | | sfGFP C terminal | | sfGFP alone | | sfGFP N terminal | | sfGFP C terminal | |
| 0.4 | 0 hour |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O/N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

Table 2. Summary of SDS-PAGE results. In each construct, the left well of the table represents the insoluble fraction and the right well the soluble fraction. Results are colour coded as follows: (Red) no evidence of expression; (yellow) Evidence of expression; (Green) Expression and secretion.

From the results shown in both Figure 8 and summarised in Table 2, we can highlight that we only identify overexpression of the untagged and with N-terminal secretion signal sfGFP constructs. However, no overexpression is visible for the HlyA tagged sfGFP (C-terminal). These results seem to correlate with the microplate RFU results shown in Figure 6. Additionally, despite the detection of the N-terminal construct, this protein does not seem to be secreted into the supernatant.

#### QUANTITATIVE RESULTS:

Cell pellets of coloured bacteria:

From the bacterial samples 5 mL were retrieved: before induction, every hour for 4 hours and overnight. Cell pellets were obtained through the centrifugation of the bacterial culture at 10,000 xg for 10 minutes. The supernatant was removed and cell pellets were transferred in the remaining media into a 96 well clear bottom microplate as seen below (Figure 6).

![](https://static.igem.org/mediawiki/2019/d/da/T--Manchester--Colours--GFPPellet.png)

Figure 9. Bacterial cell pellets from a 5 mL culture placed on a 96 well plate with transparent bottom. Cell pellets were obtained at different time points: where time 0 indicates a cell pellet at the desired OD600 of ~0.6 before induction. Time 1 h, 2 h , 3 h and 4 h show cell pellets obtained every hour for the initial 4 hours after induction with anhydrotetracycline; O/N represents the cell pellet obtained after overnight incubation at 30ºC. Plate was imaged both under normal light (A) and UV light (B) respectively for visualisation purposes only.

Visually, we can only see sfGFP production for the non-tagged (colour alone) construct. No clear colouration is seen on either of the secretion signal containing transformed bacteria. Hence, despite the detection of fluorescence in both microplate and SDS-PAGE results for the OmpA tagged construct (N-terminal), no colouration was visible to the naked eye in both normal and UV lighting. Additionally, we can highlight how the *E. coli* BL21(DE3) strain seems to be a better expression host, as expected (Rosano and Ceccarelli, 2014).

Fluorescence microscopy:

We decided to use fluorescence microscopy to check fluorescence in the cultures because the N- and C-terminal-tagged constructs were not expressing visible colour. For this, 2 µL of culture was placed on a glass slide, visualised and imaged using a fluorescence microscope (Nikon eclipse TE2000U). Results are shown below:

|  | Phase contrast (20 ms) | FP channel (600 ms) | Overlayed |
| --- | --- | --- | --- |
| Negative control (TOP10) | ![](https://static.igem.org/mediawiki/2019/6/66/T--Manchester--Colours--FM--NegP.png) | ![](https://static.igem.org/mediawiki/2019/2/23/T--Manchester--Colours--FM--NegG.png) | ![](https://static.igem.org/mediawiki/2019/2/23/T--Manchester--Colours--FM--NegG.png) |
| sfGFP alone DH5a | ![](https://static.igem.org/mediawiki/2019/3/3b/T--Manchester--Colours--FM--CA1P.png) | ![](https://static.igem.org/mediawiki/2019/5/59/T--Manchester--Colours--FM--CA1G.png) | ![](https://static.igem.org/mediawiki/2019/b/ba/T--Manchester--Colours--FM--CA1O.png) |
| sfGFP alone BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/0/03/T--Manchester--Colours--FM--CA2P.png) | ![](https://static.igem.org/mediawiki/2019/0/0d/T--Manchester--Colours--FM--CA2G.png) | ![](https://static.igem.org/mediawiki/2019/d/d9/T--Manchester--Colours--FM--CA2O.png) |
| sfGFP N-terminal DH5a | ![](https://static.igem.org/mediawiki/2019/d/db/T--Manchester--Colours--FM--NT1P.png) | ![](https://static.igem.org/mediawiki/2019/c/c9/T--Manchester--Colours--FM--NT1G.png) | ![](https://static.igem.org/mediawiki/2019/1/13/T--Manchester--Colours--FM--NT1O.png) |
| sfGFP N-terminal BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/2/20/T--Manchester--Colours--FM--NT2P.png) | ![](https://static.igem.org/mediawiki/2019/b/b3/T--Manchester--Colours--FM--NT2G.png) | ![](https://static.igem.org/mediawiki/2019/7/7f/T--Manchester--Colours--FM--NT2O.png) |
| sfGFP C-terminal DH5a | ![](https://static.igem.org/mediawiki/2019/a/ae/T--Manchester--Colours--FM--CT1P.png) | ![](https://static.igem.org/mediawiki/2019/1/17/T--Manchester--Colours--FM--CT1G.png) | ![](https://static.igem.org/mediawiki/2019/6/63/T--Manchester--Colours--FM--CT1O.png) |
| sfGFP C-terminal BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/b/b6/T--Manchester--Colours--FM--CT2P.png) | ![](https://static.igem.org/mediawiki/2019/f/f2/T--Manchester--Colours--FM--CT2G.png) | ![](https://static.igem.org/mediawiki/2019/c/c1/T--Manchester--Colours--FM--CT2O.png) |

Figure 10. Figure 10. Results obtained when using 2 µLof overnight culture during fluorescence microscopy (Nikon eclipse TE2000U). The colour alone constructs show much higher fluorescence intensity compared to N-Terminal and C-Terminal, these latter constructs show some level of background noise, but compared to the negative control there is visible amounts of GFP fluorescence. Phase images were produced at 20 ms and GFP at 600 ms exposure time. Images were processed using ImageJ.

Fluorescence microscopy test shows how colour alone construct is able to produce sfGFP and express fluorescence. The N-Terminal and C-Terminal secretion signal constructs show very limited colour, and can be seen aggregating in the poles of the cells. This suggests that colour is being produced but in limited amounts, possibly becoming a burden for the cells. A negative control (TOP10) is also presented (first row of the table above) showing no fluorescence as the TOP10.

It is important to highlight how the C-Terminal construct did not show notable fluorescence during the microplate reader experiment (Figure 6). However, when analysed under the fluorescence microscope, it was clear that it was producing some sfGFP (Figure 10). Hence, a functional GFP had to have been expressed. However, when we then looked at the different reading frames we were still unable to confirm a coding sequence.

#### CONCLUSION:

Our sets of experiments allowed us to produce and secrete the sfGFP alone construct. Therefore, through our SDS-PAGE and qualitative data, we confirmed what was suggested in the literature, that sfGFP chromoprotein is able to **auto secrete** across the membrane..

As for the N-terminal (OmpA-containing) construct, expression was achieved and demonstrated both quantitatively and qualitatively but it was not being secreted as seen by our SDS-PAGE results (Figure 8 and Table 2). Additionally, some aggregation can be seen in fluorescence microscopy experiments (Figure 10). Further experiments would have to be carried out to confirm if agregation is indeed the problem that these cells are experiencing.

Lastly, the experiments performed with the mutated sfGFP C-terminal construct [see note at the top for a more detailed explanation] showed that surprisingly, this aberrant coding sequence was producing a green chromophore. As described above, we suggest that it is working through a different Open Reading Frame (ORF); however, it would require a Western blot to prove. Nevertheless we have shown that this construct, a shorter version of sfGFP, does produce green fluorescence (Figure 10). Unfortunately, This chromoprotein was unable to be secreted across the bacterial membrane. We believe that further experiments would need to be performed in order to confirm the sequence of the protein and determine whether we have produced a minimal sfGFP.

#### Scene 2: mRFP1

mRFP1 or monomeric RFP (also referred to EngRFP) is a red fluorescent protein which was first published in 2002 by Campbell et al. . Despite mRFP1 being a monomer, it was derived from the dimeric DsRed (from Discosoma sp.) following multiple mutations. Although mRFP1 has a lower extinction coefficient, quantum yield, and photostability than DsRed, its **rapid maturation** (>10 times faster) shows similar brightness in living cells. Therefore, unlike for sfGFP and AmilCP, mRFP1 was not selected by its extinction coefficient value but instead because we believed that this monomeric chromophore would have a higher chance of correct folding and transport across the bacterial membrane.

Table 3. mRFP1 constructs. Colour alone is a previously iGEM registered part which we used as both a positive control and for further characterisation ([Contribution, Act III: Results)](https://2019.igem.org/Team%3AManchester/Contribution)). N-terminal ([BBa\_K2906005](http://parts.igem.org/Part%3ABBa_K2906005)) contains a hydrophobic tag designed for hair attachment as well as an N-terminal signal sequence required for secretion; similarly C-terminal ([BBa\_K2906006](http://parts.igem.org/Part%3ABBa_K2906006)) follows the same structure however in this case the signal required for secretion is instead located on the C-terminal region.

| Name | Registry Code | BioBrick |
| --- | --- | --- |
| Colour alone | BBa\_K092300 previously existing iGEM part | ![](https://static.igem.org/mediawiki/2019/1/11/T--Manchester--Colours--RFP1.png) |
| N-terminal | BBa\_K2906005 | ![](https://static.igem.org/mediawiki/2019/5/59/T--Manchester--Colours--RFP2.png) |
| C-terminal | BBa\_K2906006 | ![](https://static.igem.org/mediawiki/2019/4/48/T--Manchester--Colours--RFP3.png) |

For the characterisation of mRFP1 constructs, the same procedure as with sfGFP was undertaken. However, negative controls in this case included uninduced pBbB2c-GFP in both DH5⍺ and BL21(DE3) respectively, as well as the *E. coli* TOP10 strain without vector.

#### QUANTITATIVE RESULTS:

Characterisation using microplate reader:

Measurements were obtained as in sfGFP however in this case the microplate reader was set to the RFU mRFP1 protocol. Below are the results we obtained for the OD and fluorescence measurements of the different constructs with mRFP1. All the values were analysed by blank-correction. Optical Density values were measured at 660 nm, this was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria. For OD blank was LB media, and for RFU the blank was *E. coli* TOP10 since it does not express any colour. The values were normalised by dividing RFU/OD, and then averaged to plot the mean against time. An RFU value of 0 corresponds to baseline *E. coli* TOP10 measurements.

Characterisation on microplate reader:

OD660

![](https://static.igem.org/mediawiki/2019/1/14/T--Manchester--Colours--RFPGeneral.png)

Figure 11. The plot shows the mean RFU/OD from three replicates of each construct expressed in *E. coli* DH5⍺ and BL21(DE3) respectively. The OD was measured at 660 nm and RFP fluorescence was measured at Ex ƛ 574, Em ƛ 618, every 15 minutes for 13 hours. The RFU values were normalised by the OD and the triplicates averaged. All values have been blank-corrected by LB (OD) and *E. coli* TOP10 (RFU), therefore an RFU/OD value of 0 is equivalent to TOP10 fluorescence (negative control). A total of 52 recordings were made per well, with three wells per construct.

From our normalised data we can observe that the colour alone construct produces a higher fluorescence with respect to the other two designed parts. Also, the C-terminal secretion signal part is indeed produced and detected. However, the N-terminal construct does not appear to be produced. Bacterial growth - determined by looking at OD660 - is normal for all 3 mRFP1 construct variants (data not shown). Hence, the lack of colouration from the N-terminal construct does not seem to interfere with bacterial growth i.e. it is not toxic to the cells. Nevertheless, mRFP1 from the N-terminal construct does not seem to be produced, folded or secreted altogether.

SDS-PAGE:

A 5 mL sample was obtained both before induction (time 0) and after induction and incubation at 30ºC. The expected molecular weight was determined: mRFP1 alone is 25.4 kDa, with the N-terminal tag it is 27.6 kDa (26.1 KDa when fully cleaved and secreted) and with the C-terminal tag it is 31.6 kDa.

![](https://static.igem.org/mediawiki/2019/1/13/T--Manchester--Colours--NOBGRFPSDS1.png)

![](https://static.igem.org/mediawiki/2019/6/60/T--Manchester--Colours--NOBGRFPSDS2.png)

![](https://static.igem.org/mediawiki/2019/0/02/T--Manchester--Colours--NOBGRFPSDS3.png)

![](https://static.igem.org/mediawiki/2019/8/89/T--Manchester--Colours--NOBGRFPSDS4.png)

![](https://static.igem.org/mediawiki/2019/b/b9/T--Manchester--Colours--NOBGRFPSDS5.png)

![](https://static.igem.org/mediawiki/2019/d/dc/T--Manchester--Colours--NOBGRFPSDS6.png)

Figure 12. SDS-PAGE gels (Mini-PROTEAN TGX Stain-Free Precast gels 10%). A 5 mL sample was taken from each construct before induction (negative control) and after overnight inoculation at 30ºC following induction with anhydrotetracycline (O/N). The expected molecular sizes: Colour alone (26.8 kDa); N-terminal (29.2 kDa or 27.7 when fully cleaved and secreted) and C-terminal of 33.2 kDa. All samples were checked for protein expression. Supernatant, soluble and insoluble fractions were all analysed during this experiment.

The gels have some background noise, suggesting that there might be cells present in the supernatant. To overcome this we filter-sterilised the supernatant (0.2-micron filter). This additional sterilisation step was only carried out for the C-terminal construct.

![](https://static.igem.org/mediawiki/2019/0/08/T--Manchester--Colours--NOBGRFPSDS7.png)

Figure 13. SDS-PAGE of filtered-sterilised samples to verify secretion of the desired protein. Expected molecular weight of secreted mRFP1 C-terminal construct is 31.6 kDa. Negative control is provided in Figure 23.

The gel confirms that mRFP1 C-terminal is not only **overexpression**but also being secreted into the supernatant.

![](https://static.igem.org/mediawiki/2019/6/65/T--Manchester--TABLE.png)

Table 4. Summary of SDS-PAGE results. In each construct, the left well of the table represents the insoluble fraction and the right well the soluble fraction. (Red) no evidence of expression; (yellow) Evidence of only expression; (Green) Expression and secretion; (Pink) Secretion verified by filter sterilizing supernatant to remove potential cells. Alternative colour alone constructs not shown in this table.

Our SDS-PAGE results indicate that both Colour Alone and C-terminal constructs overexpress mRFP1 under the growth conditions provided when induced. However, the N-terminal construct was unable to produce the chromophore. Additionally, the C-terminal construct was also shown to be secreted into the supernatant.

#### QUALITATIVE RESULTS:

Cell pellets of coloured bacteria:

![](https://static.igem.org/mediawiki/2019/1/16/T--Manchester--Colours--RFPPellets.png)

Figure 14. 5 mL bacterial cell pellets placed on a 96-well plate with transparent bottom. cell pellets were obtained at different time points: where time 0 indicates a cell pellet at the desired OD600 of ~0.6 before induction, time 1 h, 2 h , 3 h and 4 h show cell pellets obtained every hour for the initial 4 hours after induction with anhydrotetracycline; O/N represents the cell pellet obtained after overnight incubation at 30ºC. Plate was imaged both under normal light.

The figure above allows us to visualise colour production of the colour alone and C-terminal secretion signal constructs respectively. However, no colouration is seen in the N-terminal secretion signal construct which correlates with the results obtained using the microplate reader and SDS-PAGE respectively.

Fluorescence microscopy:

We decided to use fluorescence microscopy to check fluorescence in the cultures because they were **not expressing visible colour**. 2 µL of culture was placed on a glass slide, visualised and imaged using a fluorescence microscope. Results are shown below:

|  | Phase contrast (20 ms) | FP channel (600 ms) | Overlayed |
| --- | --- | --- | --- |
| Negative control (TOP10) | ![](https://static.igem.org/mediawiki/2019/7/70/T--Manchester--Colours--FMRFP--NegP.png) | ![](https://static.igem.org/mediawiki/2019/7/7c/T--Manchester--Colours--FMRFP--NegR.png) | ![](https://static.igem.org/mediawiki/2019/3/31/T--Manchester--Colours--FMRFP--NegO.png) |
| mRFP1 alone DH5a | ![](https://static.igem.org/mediawiki/2019/1/16/T--Manchester--Colours--FMRFP--CA1P.png) | ![](https://static.igem.org/mediawiki/2019/8/86/T--Manchester--Colours--FMrfp--CA1R.png) | ![](https://static.igem.org/mediawiki/2019/e/ed/T--Manchester--Colours--FMrfp--CA1O.png) |
| mRFP1 alone BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/f/f4/T--Manchester--Colours--FMrfp--CA2P.png) | ![](https://static.igem.org/mediawiki/2019/f/f0/T--Manchester--Colours--FMrfp--CA2R.png) | ![](https://static.igem.org/mediawiki/2019/0/0a/T--Manchester--Colours--FMrfp--CA2O.png) |
| mRFP1 N-terminal DH5a | ![](https://static.igem.org/mediawiki/2019/7/7e/T--Manchester--Colours--FMrfp--NT1P.png) | ![](https://static.igem.org/mediawiki/2019/0/03/T--Manchester--Colours--FMrfp--NT1R.png) | ![](https://static.igem.org/mediawiki/2019/7/7d/T--Manchester--Colours--FMrfp--NT1O.png) |
| mRFP1 N-terminal BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/a/ad/T--Manchester--Colours--FMrfp--NT2P.png) | ![](https://static.igem.org/mediawiki/2019/2/22/T--Manchester--Colours--FMrfp--NT2.png) | ![](https://static.igem.org/mediawiki/2019/f/fb/T--Manchester--Colours--FMrfp--NT2O.png) |
| mRFP1 C-terminal DH5a | ![](https://static.igem.org/mediawiki/2019/d/dd/T--Manchester--Colours--FMrfp--CT1P.png) | ![](https://static.igem.org/mediawiki/2019/4/45/T--Manchester--Colours--FMrfp--CT1R.png) | ![](https://static.igem.org/mediawiki/2019/c/c8/T--Manchester--Colours--FMrfp--CT1O.png) |
| mRFP1 C-terminal BL21(DE3) | ![](https://static.igem.org/mediawiki/2019/3/34/T--Manchester--Colours--FMrfp--CT2P.png) | ![](https://static.igem.org/mediawiki/2019/f/fa/T--Manchester--Colours--FMrfp--CT2R.png) | ![](https://static.igem.org/mediawiki/2019/0/0c/T--Manchester--Colours--FMrfp--CT2O.png) |

Figure 15. Results obtained when using 2 µLof overnight culture during fluorescence microscopy (Nikon eclipse TE2000U). Phase construct produced at 20 ms and GFP at 600 ms .Images were processed using ImageJ.

Similarly to our previous results, this fluorescent microscopy test shows how both the colour alone and C-terminal secretion construct are able to produce mRFP1 and express fluorescence. Though the N-terminal secretion signal shows mostly background under the fluorescence filter, there are a number of colonies expressing mRFP1 in BL21 cells. Hence, unlike with our previous results, this fluorescence microscopy test did indeed show us how this N-terminal construct was able to express the chromoprotein, yet it was not visible with the naked eye. A negative control is also presented (first row of the table above) showing no fluorescence as the TOP10 was not expected to fluoresce.

#### CONCLUSION:

Despite the lack of production of the N-terminal secretion signal mRFP1 variant, both colour alone an C-terminal constructs respectively were successfully produced. Additionally the novel HylA tagged-mRFP1 containing a hydrophobic tag was successfully produced and to the best of our knowledge found secreted in the supernatant of the bacterial culture (this was further tested through filter-sterilising the supernatant during SDS-PAGE preparation). Hence, making mRFP1 C-terminal construct a good option for our final product in future experiments. Additionally, improvements into the secretion systems could be performed in the future- overexpression of the transporters or relevant chaperones could allow improvements on secretion.

#### Scene 3: amilCP

AmilCP, from the coral Acropora millepora, in contrast to GFP and RFP, is a chromoprotein which is **not fluorescent**. We have specifically chosen this chromoprotein to show the versatility of our approach - merely limited by the intensity of the color (modelling). Furthermore, we picked AmilCP because its characterization outside of iGEM is limited - we wanted to explore this interesting blue chromoprotein in more detail

Table 5. AmilCP constructs. Colour alone [BBa\_K1455001](http://parts.igem.org/Part%3ABBa_K1455001)is a previously iGEM registered part which we used as a positive control and for further characterisation. N-terminal ([BBa\_K2906003](http://parts.igem.org/Part%3ABBa_K2906003)) contains a hydrophobic tag designed for hair attachment as well as an N-terminal signal sequence required for secretion; similarly C-terminal ([BBa\_K2906004](http://parts.igem.org/Part%3ABBa_K2906004)) follows the same structure, however in this case the signal required for secretion is located on the C-terminal region.

| Name | Registry Code | BioBrick |
| --- | --- | --- |
| Colour alone | BBa\_K1455001 previously existing iGEM part | ![](https://static.igem.org/mediawiki/2019/3/33/T--Manchester--Colours--AmilCP1.png) |
| N-terminal | BBa\_K2906003 | ![](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--Colours--AMilCP2.png) |
| C-terminal | BBa\_K2906004 | ![](https://static.igem.org/mediawiki/2019/0/0f/T--Manchester--Colours--AmilCP3.png) |

The same protocol for growing the cultures was undertaken as for the fluorescent chromophores. However, in this case the selected negative control was pBbB2c-GFP transformed into DH5⍺ cells which was not induced. This experiment was repeated twice, in order to have more data to characterise and validate the **absorbance maxima** of AmilCP. Our initial experiment (replicate 1) was performed by induction of *E. coli* cultures at OD600 = ~0.4. We later performed the same experimental procedure (replicate 2) with induction at OD600 = ~0.6. Once the desired OD600 was reached, a 0h sample was taken: 1 mL was used to measure both

1. absorbance at 588 nm,

2. OD600, and

3. whole spectra analysis.

Additionally, cell pellets were obtained by centrifugation of 5 mL culture at 10,000 xg, then cell pellets were transferred to individual wells of a clear 96-well plate (Figure 24). 5 mL of sample was also obtained for the SDS-PAGE analysis. Samples were then induced. Every hour for four hours after induction, 1 mL was obtained for a spectrophotometer scan (including whole spectra, specific 588 nm reading and OD600 for monitoring of growth) and 5 mL bacterial cell pellet was obtained (following the same procedure explained above) and placed on a 96-well plate (see qualitative data). Lastly, after overnight incubation at 30ºC, a 5 mL sample was retrieved for SDS-PAGE and an additional 5 mL cell pellet was obtained as above.

#### QUANTITATIVE RESULTS:

Characterisation using spectrophotometer:

Once the desired OD600 had been obtained (0.4 for replicate 1 and 0.6 for replicate 2) an initial 1 mL of sample from each bacterial culture was obtained. These samples were then analysed using a spectrophotometer. The spectrophotometer was used to perform: a whole spectra analysis (Figure 16-22); an OD600 reading (Figure 25-26) and an OD588 readings (Figure 23-24).

Then samples were induced with 100 nM of anhydrotetracycline (with the exception of negative control pBbB2c-GFP *E. coli* DH5⍺). The flasks were placed at 30ºC with shaking at 180 rpm. Measurements were performed using a spectrophotometer (Cary 60, Agilent Technologies) every hour for 4 hours and overnight.

For OD measurements, the blank was LB media, and for absorbance the blank was uninduced pBbB2c-GFP which does not express any colour as TetR is not a leaky promoter. However, in the unlikely attempt of colour production, it would not be in the same range as AmilCP and hence it would not be detected by these sets of experiments. Then, specific absorbance measurements at discrete wavelengths was performed. This was done at 600 nm to measure optical density and bacterial growth, and at 588 nm because it has been previously shown to be peak absorbance of AmilCP. For all figures, individual measurements were plotted. Additionally, we decided to perform a full spectrum analysis for each construct in order to measure the maximum absorbance (Figure 19. summarising results). All the values plotted were previously processed by blank-correction.

Results below show figures from analysed data of the spectrophotometer full-spectrum readings.

![](https://static.igem.org/mediawiki/2019/thumb/0/01/T--Manchester--Colour-CAO4-1.png/800px-T--Manchester--Colour-CAO4-1.png)
![](https://static.igem.org/mediawiki/2019/thumb/2/2d/T--Manchester--Colour-CAO4-A.png/800px-T--Manchester--Colour-CAO4-A.png)

![](https://static.igem.org/mediawiki/2019/thumb/0/02/T--Manchester--Colour-CAO4-2.png/800px-T--Manchester--Colour-CAO4-2.png)
![](https://static.igem.org/mediawiki/2019/thumb/2/2d/T--Manchester--Colour-CAO4-A.png/800px-T--Manchester--Colour-CAO4-A.png)

Figure 16. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. Local maxima around 580-600 nm shows excess AmilCP production. DH5⍺ cells were more pronounced than BL21(DE3), however in both plots it is clear that there is a mean peak at 582.5 nm (overnight values).

![](https://static.igem.org/mediawiki/2019/thumb/e/ea/T--Manchester--Colour-NTO4-1.png/800px-T--Manchester--Colour-NTO4-1.png)
![](https://static.igem.org/mediawiki/2019/thumb/b/b2/T--Manchester--Colour-NTO4-A.png/800px-T--Manchester--Colour-NTO4-A.png)

![](https://static.igem.org/mediawiki/2019/thumb/c/cc/T--Manchester--Colour-NTO4-2.png/800px-T--Manchester--Colour-NTO4-2.png)
![](https://static.igem.org/mediawiki/2019/thumb/b/b2/T--Manchester--Colour-NTO4-A.png/800px-T--Manchester--Colour-NTO4-A.png)

Figure 17. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. A lack of a local maxima around 580-600 nm shows that there is only a minimal production or that the fusion protein may not fold correctly. Bacterial cultures seem to be growing slower suggesting possible aggregation or misfolding of the protein.

![](https://static.igem.org/mediawiki/2019/thumb/c/c1/T--Manchester--Colour-CTO4-1n.png/800px-T--Manchester--Colour-CTO4-1n.png)
![](https://static.igem.org/mediawiki/2019/thumb/6/6c/T--Manchester--Colour-CTO4-An.png/800px-T--Manchester--Colour-CTO4-An.png)

![](https://static.igem.org/mediawiki/2019/thumb/3/3b/T--Manchester--Colour-CTO4-2n.png/800px-T--Manchester--Colour-CTO4-2n.png)
![](https://static.igem.org/mediawiki/2019/thumb/6/6c/T--Manchester--Colour-CTO4-An.png/800px-T--Manchester--Colour-CTO4-An.png)

Figure 18. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. local maxima around 580-600 nm shows that there is some AmilCP production. The arrow in the first plot shows absorbance maxima, while the second plot did not show any clear peaks.

![](https://static.igem.org/mediawiki/2019/thumb/c/ca/T--Manchester--Colour-CAO6-1.png/800px-T--Manchester--Colour-CAO6-1.png)
![](https://static.igem.org/mediawiki/2019/thumb/c/cf/T--Manchester--Colour-CAO6-A.png/800px-T--Manchester--Colour-CAO6-A.png)

![](https://static.igem.org/mediawiki/2019/thumb/4/4f/T--Manchester--Colour-CAO6-2.png/800px-T--Manchester--Colour-CAO6-2.png)
![](https://static.igem.org/mediawiki/2019/thumb/c/cf/T--Manchester--Colour-CAO6-A.png/800px-T--Manchester--Colour-CAO6-A.png)

Figure 19. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. local maxima around 580-600 nm shows that there is AmilCP production. Arrows on both plots show the clear absorbance maxima for AmilCP. The mean peak between both plots is 587.5 nm (overnight values).

![](https://static.igem.org/mediawiki/2019/thumb/8/81/T--Manchester--Colour-NTO6-1.png/800px-T--Manchester--Colour-NTO6-1.png)
![](https://static.igem.org/mediawiki/2019/thumb/5/5e/T--Manchester--Colour-NTO6-A.png/800px-T--Manchester--Colour-NTO6-A.png)

![](https://static.igem.org/mediawiki/2019/thumb/6/60/T--Manchester--Colour-NTO6-2.png/800px-T--Manchester--Colour-NTO6-2.png)
![](https://static.igem.org/mediawiki/2019/thumb/5/5e/T--Manchester--Colour-NTO6-A.png/800px-T--Manchester--Colour-NTO6-A.png)

Figure 20. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. A lack of a local maxima around 580-600 nm shows that there is only a minimal production or that the fusion protein may not fold correctly. Bacterial cultures seem to be growing slower suggesting possible toxic metabolites.

![](https://static.igem.org/mediawiki/2019/thumb/c/c3/T--Manchester--Colour-CTO6-1.png/800px-T--Manchester--Colour-CTO6-1.png)
![](https://static.igem.org/mediawiki/2019/thumb/5/5b/T--Manchester--Colour-CTO6-A.png/800px-T--Manchester--Colour-CTO6-A.png)

![](https://static.igem.org/mediawiki/2019/thumb/4/44/T--Manchester--Colour-CTO6-2.png/800px-T--Manchester--Colour-CTO6-2.png)
![](https://static.igem.org/mediawiki/2019/thumb/5/5b/T--Manchester--Colour-CTO6-A.png/800px-T--Manchester--Colour-CTO6-A.png)

Figure 21. Whole spectra reading. Full spectrum analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. Measurements were performed at one-hour intervals for four hours, and then overnight. Some local maxima around 580-600 nm shows that there is some AmilCP production. Arrows on both plots show clear maxima for AmilCP absorption.

Processed data, overnight plots of replicate 2:

![](https://static.igem.org/mediawiki/2019/7/79/T--Manchester--Colours--OvernightScan.png)

Figure 22. Whole-spectra scans after overnight incubation of all different constructs in DH5⍺ and BL21(DE3). Analysis was performed with a spectrophotometer (Cary 60, Agilent technologies) from 300 to 800 nm. All of the overnight scans overlayed show that there is a mean absorbance peak at 588 nm (arrow).

The absorbance spectrums above spanned from 300 to 800 nm, defined by a local peak in the range of 580 and 595 nm. From the results above, we calculated the mean of obtained absorbance maxima. The value obtained was 587.5 nm and hence we can conclude that indeed the absorbance maxima for AmilCP chromoprotein is 588 nm. We also performed an extra experiment (not shown) were we measured absorbance and emittance, and this confirms that AmilCP is not a fluorescent protein despite its being a GFP-like variant.

OD:

OD600 0.4:

![](https://static.igem.org/mediawiki/2019/1/19/T--Manchester--Colours--AmOD600_0.4.png)

Figure 23. Replicate 1 (induced at OD600 of ~0.4). The plot shows the growth of bacteria over time determined by its OD600. Measurements were taken at 6 different time points: before induction (0 hour), during the 4 hour monitoring and after overnight incubation at 30ºC. The negative control was pBbB2c-GFP uninduced because it does not produce colour. Additionally, in the unlikely event that it produces GFP, this spectrum would not affect our readings from AmilCP

OD600 0.6:

![](https://static.igem.org/mediawiki/2019/d/d0/T--Manchester--Colours--AmOD600_0.6.png)

Figure 24. Replicate 2 (induced at OD600 of ~0.6). The plot shows the growth of bacteria over time determined by its OD600. Measurements were taken at 6 different time points: before induction (0 hour), during the 4 hour monitoring and after overnight incubation at 30ºC. The negative control was pBbB2c-GFP uninduced because it does not produce colour. Additionally, in the unlikely event that it produces GFP, this spectrum would not affect our readings from AmilCP

From our OD600 measurements we can clearly observe the normal growth over time of the colour alone and C-terminal constructs respectively. However, the N-terminal transformed bacteria seem to decline over time in the induced samples at OD600 ~0.4. This is true in *E. coli* DH5⍺ and BL21(DE3). While this is not equally as visible in replicate 2 (when cells were induced at OD600 of ~0.6) we can however highlight a degree of stunted growth. From the growth determination we can conclude that the AmilCP N-terminal part must be causing toxic aggregates in the inclusion bodies of *E. coli* cells. This was later confirmed by SDS-PAGE.

OD588 0.6:

![](https://static.igem.org/mediawiki/2019/4/41/T--Manchester--Colours--AmOD588_0.6.png)

Figure 25. Replicate 1. The above plot show *E. coli* absorbance at 588 nm which is the absorbance maxima of AmilCP. Measurements were made at 5 different time points. The negative control was pBbB2-GFP uninduced because it did not produce any colouration.

OD588 0.4:

![](https://static.igem.org/mediawiki/2019/7/75/T--Manchester--Colours--AmOD588_0.4.png)

Figure 26. Replicate 2. The above plot show *E. coli* absorbance at 588 nm which is the absorbance maxima of AmilCP. Measurements were made at 5 different time points. The negative control was pBbB2-GFP uninduced because it did not produce any colouration.

The above plots suggest that discrete measurements of *E. coli* cultures at 588 nm are not an accurate representation of AmilCP production in bacteria. As we can see in the spectrum scan, the same colonies could be easily shown to produce, or not to produce AmilCP. This might be because 588 nm is very close to 600 nm, the wavelength at which the optical density of *E. coli* is measured. Therefore there is interference and noise when measured at a discrete wavelength. To the best of our knowledge, the best way to measure AmilCP production is by spectral scan and seeing relative increase in the absorbance.

SDS-PAGE:

Additional to the determination of the AmilCP absorbance characteristics, we also wanted to determine if the protein was overexpressed and secreted from the bacterial cell. In order to identify this we used SDS-PAGE from 5 mL samples obtained at the desired OD600 before induction and induced after overnight incubation at 30ºC with shaking.

We initially calculated the expected sizes of AmilCP: We determined that the colour alone was expected to have a 25.4 kDa molecular weight, whereas the N-terminal tag and cleaved C-terminal tag of 27.6 kDa (or 26.4 kDa when fully cheaved) and 31.6 kDa respectively. 10% gels were run and stained with Coomassie blue, then they were scanned:

![](https://static.igem.org/mediawiki/2019/8/85/T--Manchester--Colours--AmilCPSDS1.png)

![](https://static.igem.org/mediawiki/2019/c/cc/T--Manchester--Colours--AmilCPSDS2.png)

![](https://static.igem.org/mediawiki/2019/e/e0/T--Manchester--Colours--AmilCPSDS3.png)

![](https://static.igem.org/mediawiki/2019/6/67/T--Manchester--Colours--AmilCPSDS4.png)

Figure 27. SDS-PAGE gels (Mini-PROTEAN TGX Stain-Free Precast gels 10%). A 5 mL sample was taken from each construct before induction (negative control) and after overnight inoculation at 30ºC following induction with anhydrotetracycline (O/N). The expected molecular sizes: Colour alone (25.4 kDa); N-terminal (27.6 kDa or 26.4 when fully cleaved and secreted) and C-terminal of 31.6 kDa. All samples were checked for protein expression. Supernatant, soluble and insoluble fractions were all analysed during this experiment.

The gels for the supernatant above show some background, which means that some cells could be present. To overcome this, and to prove secretion of our construct, we decided to filter-sterilise the supernatant by passing it through a 0.2-micron filter. Then, these samples were prepared for SDS-PAGE. Note that this additional step was only undertaken with samples from replicate 1 (Figure 23).

![](https://static.igem.org/mediawiki/2019/d/dd/T--Manchester--Colours--AmilCPSDS5.png)

Figure 28. SDS-PAGE of filtered-sterilised samples to verify secretion of the desired protein. Expected molecular weight: AmilCP alone (25.4), AmilCP N-terminal (26.4) and C-terminal (31.6). Negative control is provided in Figure 13.

The following Table summarises the obtained SDS-PAGE results.

Table 6. Summary of SDS-PAGE results. In each construct, the left well of the table represents the insoluble fraction and the right well the soluble fraction. Results are colour coded as follows: (Red) no evidence of expression; (yellow) Evidence of expression; (Green) Expression and secretion; (Pink) Secretion verified by filter sterilizing supernatant to remove potential cells.

|  |  | DH5⍺ | | | | | | | | BL21(DE3) | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Negative control | | AmilCP alone | | AmilCP N terminal | | AmilCP C terminal | | AmilCP alone | | AmilCP N terminal | | AmilCPC terminal | |
| 0.4 | 0 hour |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O/N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| 0.6 | 0 hour |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O/N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

Based on our SDS-PAGE results we can conclude that AmilCP overexpression was only obtained in two of the three designed constructs, in both the E coli strains used. Whereas expression is readily apparent in both the colour alone and C-terminal construct; the N-terminal expression is only obtained in replicate 1 when cells were induced at an OD600 of 0.4. Additionally, from our results we can determine that unexpectedly the Colour Alone (AmilCP Alone) construct was not only overexpressed under the exposed growth conditions but it was also able to auto secrete out of the cell membrane in both *E. coli* DH5⍺ and BL21(DE3). On the other hand, despite apparent expression of AmilCP with C-terminal secretion signal, this construct was unable to be secreted.

#### QUALITATIVE RESULTS:

Cell pellets of coloured bacteria:

Similarly to sfGFP, 5 mL bacterial cell pellets were produced before induction and every hour following induction for 4 hours, as well as an overnight.

![](https://static.igem.org/mediawiki/2019/8/88/T--Manchester--Colours--AmPellets.png)

Figure 29. 5 mL bacterial cell pellets placed on a 96-well plate with transparent bottom. At each time point, the right column shows replicate 1 (induced at OD600 of 0.4) and the left column shows replicate 2 (induced at OD600 of 0.6).

Similarly to our SDS-PAGE results, we can visually observe high AmilCP production of the colour alone and C-terminal construct; whereas no apparent colouration is seen in the AmilCP N-terminal transformed cells. Additionally, these results seem to suggest less overproduction of AmilCP in the C-terminal construct, whereas when analysing the SDS-PAGE gels this does not seem to correlate. Hence, this observation seems to indicate that the AmilCP C-terminal production is unable to be secreted because of aberrant folding, which could also explain the reduction of visible colour with respect to the non-tagged (colour alone) construct. As for the N-terminal constructs, a smaller non-coloured cell pellet was detected and this seems to correlate with the obtained OD600 and whole spectra results. Overproduction and the formation of aggregates could ultimately be toxic for bacteria themselves. We cannot detect any visual difference in colour expression between *E. coli* DH5⍺ and BL21(DE3) respectively.

#### CONCLUSION:

The sets of performed experiments allowed us to verify that the absorbance maxima of this blue chromoprotein is indeed 588 nm. The analysis of SDS-PAGE results after expression indicate that, unexpectedly, AmilCP could be secreted through the membrane of both *E. coli* DH5⍺ and BL21(DE3), but if secretion is a passive or active process in this case needs further investigation. Suspected aggregation inhibited the correct folding of AmilCP N-terminal and hence this protein despite being produced (as seen by our SDS-results, Figure 23 and Table 6) did not produce the expected intense colour (Qualitative data, Figure 24) or become secreted across the membrane. Additionally, AmilCP N-terminal production was not obtained when induction was performed at an OD600 of 0.6 probably due to the formation of those toxic aggregates that inhibited growth altogether. Lastly, AmilCP C-terminal (HlyA tagged) was seen to produce colouration both through our spectra analysis (Figure 21-22) as well as qualitatively as it is seen in pellets with the naked eye in normal light (Figure 24). However, our SDS-PAGE results (Figure 23 and summarised in Table 6) seem to suggest that this construct is not able to become secreted. We therefore hypothesised that aberrant folding could be taking place causing the inhibition of the chromophore ́s secretion

For the above, we show that structural changes to the protein could aid and allow the secretion of the chromoprotein in future experiments.

#### Act III: Applications

“Swallow the sunset and drink the rainbow” - Khalil Gibran

**Are we limited? No, we have a wide range of colours!**

During the summer months doing the project, we contacted various **stakegolders** to get an industrial insight, get feedback, share our ideas, and learn all that we could from experienced people. One of the most important meetings we had was with Seamus McCrory, the owner of McCrory Hairdressers. He is an expert in colouring hair and he said, amongst many things, that we had to be competitive and offer a wide range of colours, equalling if not surpassing chemical hair dyes in variety. Therefore, we decided to combine our different constructs and extend our colour palette. We combined different concentrations of cell pellets expressing our colourful proteins, to create gradients of colour that look different in white visible and UV light. Additionally, we proved that bacteria with our construct **attach to hair** making them feasible as a hair dye.

![](https://static.igem.org/mediawiki/2019/b/be/T--Manchester--Colours--ColourRange.png)

Figure 30. Palette of colours obtained by mixing the three primary chromophores in Cutiful, shown absorbing in the visible spectrum.

![](https://static.igem.org/mediawiki/2019/c/c1/T--Manchester--Colours--ColourRangeUV.png)

Figure 31. Palette of colours obtained by mixing the three primary chromophores in Cutiful, shown emitting under UV.

#### Act IV: Overall Conclusions

“There is no blue without yellow and orange” - Vincent Van Gogh

From this set of experiments we have been able to achieve successful cloning of 7 newly designed iGEM constructs and 2 additional previously registered iGEM parts. These constructs were designed with the aim of producing a protein-based hair dye alternative to options currently on the market. Hence, we adapted a hydrophobic tag in order to allow interaction between the bacterial biosynthesised chromoprotein and the outer layer of the cuticle of hair (which is characterised to be highly hydrophobic). Additionally, the un-tagged constructs were also tested, as experiments regarding hair adhesion proved that the *E. coli* strains that we were using were able to naturally attach to hair.

We also designed and characterised a new, essential, composite part: tet + sfGFP. This will allow future iGEM teams to use this part for several, varied projects. This part was additionally seen to be an improvement compared to available iGEM registered parts ([Improvement, Act III: Results](https://2019.igem.org/Team%3AManchester/Improve)). We also verified and further characterised the untagged version of the blue chromoprotein AmilCP, showing that it indeed it has an absorption maxima of 588 nm. This will aid future usage of the chromoprotein by other teams as it will provide an accurate set-point for characterisation. We additionally identify how whole spectra analysis is the best way of determining this proteins expression.

Moreover, we characterised a new functional part, mRFP1 C-Terminal ([BBa\_K2906006](http://parts.igem.org/Part%3ABBa_K2906006)), proved through quantitative and qualitative experiments. Finally, we provided standardised data to previous iGEM-registered parts (further characterisation found in the characterisation tab).

Lastly, we tested an N-terminal secretion signal part variant. Despite this part not being functional we believe that further experiments could improve problems regarding folding and or trafficking which could be inhibiting the production of this chromophore.

#### Act V: Future Perspectives for Colour in Cutiful

“I made them sing with all the intensity I could.” - Wassily Kandinsky

From our initial design and modelling we have been able to achieve a **wide range of colours** and have demonstrated that through the use of bacterial co-cultures. Hence, our dye could work similarly to paint, in which different ratios could result in a different colour being produced. Due to time constraints most of these colours would however still remain inside the bacteria, and whilst we have also demonstrated that this would achieve hair colouring ([Demonstration Act II: Results](https://2019.igem.org/Team%3AManchester/Demonstrate)), future experiments working on successful secretion and folding of our selected chromophores would achieve a much more versatile end product.

In the future we would focus on showing the attachment of the chromoprotein to hair and the characterisation of our novel adapted hydrophobic tag. We would also like to measure colour retention in virgin and bleached hair respectively and measure how the different chromophores interact with hair when applied directly.

These sets of experiments could also feed into new branches of the hair dye industry and provide more colours for educational kits such as those proposed by Stark et al.

##### References

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# Team:Manchester/Results

UoM iGEM | Project Cutiful

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* PROJECT

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  ##### Description

  Brief Overview of Project](https://2019.igem.org/Team%3AManchester/Description)
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  ##### Modelling

  Theory and Aims](https://2019.igem.org/Team%3AManchester/Model)
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  ##### Timeline

  Chronological Project Layout](https://2019.igem.org/Team%3AManchester/Notebook)
* EXPERIMENT

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  ##### Colour

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Experiments)
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  ##### Fragrance

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Fragrance)
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  ##### Repair

  Protocols and Results](https://2019.igem.org/Team%3AManchester/Repair)
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  ##### Demonstration

  Our work in real life](https://2019.igem.org/Team%3AManchester/Demonstrate)
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  ##### Safety

  In the Lab, and in Our Project](https://2019.igem.org/Team%3AManchester/Safety)
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  ##### Measurement

  Special Prize](https://2019.igem.org/Team%3AManchester/Measurement)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Protocols

  Our protocols](https://2019.igem.org/Team%3AManchester/Protocols)
* PARTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Parts Collection

  Our New Biobricks](https://2019.igem.org/Team%3AManchester/Part_Collection)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Contribution

  Chracterization of Existing Biobricks](https://2019.igem.org/Team%3AManchester/Contribution)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Improvement

  Our Improved Biobricks](https://2019.igem.org/Team%3AManchester/Improve)
* HUMAN PRACTICES

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Integrated Human Practices

  Cutiful in the Wider world](https://2019.igem.org/Team%3AManchester/Human_Practices)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Public Engagement

  Protocols and results for colour](https://2019.igem.org/Team%3AManchester/Public_Engagement)
* TEAM

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Members

  Meet the Team](https://2019.igem.org/Team%3AManchester/Team)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Attributions

  Our Advisors, Contributors and Sponsors](https://2019.igem.org/Team%3AManchester/Attributions)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Collaborations

  Outreach Within iGEM Teams](https://2019.igem.org/Team%3AManchester/Collaborations)
* ACHIEVEMENTS

  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Medal Criteria

  Where We Impress You](https://2019.igem.org/Team%3AManchester/Medal)
  [![icon-1](https://static.igem.org/mediawiki/2019/4/40/T--Manchester--nav-d-icon.png)

  ##### Judging Form

  At the Judges' Convenience](https://igem.org/2019_Judging_Form?team=Manchester)
* [![](https://static.igem.org/mediawiki/2019/archive/4/4c/20191005093133%21T--Manchester--tw-icon.png)](https://twitter.com/iGEM_Manchester)
  [![](https://static.igem.org/mediawiki/2019/archive/0/0f/20191005093741%21T--Manchester--ig-icon.png)](https://www.instagram.com/igem.manchester/)

![](https://static.igem.org/mediawiki/2019/thumb/3/3d/T--Manchester--Template-BackArrow.png/800px-T--Manchester--Template-BackArrow.png)

# Results

Here you can describe the results of your project and your future plans.

### 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.** 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)