UoM iGEM | Project Cutiful

PARTS COLLECTION

This is where we dazzle! Improved Parts, Validated Parts, Characterised Parts - they are all here for your perusal. From Gold #2 to Silver #1 and Bronze #5: if there is one thing you must take from this - it is that we’ve worked, we’ve worked hard - and our experiments too!

Our Achievements …

In 3 bullet points

1.

Gold#2 Improved Part- our part, BBa_K2906000 (pTet+sfGFP), is an improvement of BioBrick: BBa_I746909, distributed out in iGEM kits.

2.

Silver#1 New Functional Part - we have four new parts: BBa_K2906006 (pTet+hydrophobic tag + mRFP1 + C-terminal secretion signal (HlyA)), BBa_K2906012 (T7 promoter- RBS- Sam8- T7 terminator), BBa_K2906014 (T7 promoter- RBS- Sam5- T7 terminator), and BBa_K2906016 (T7 promoter- RBS- COMT- T7 terminator).

3.

Bronze#5 Characterising Existent Part - we characterised two already existent biobricks: BBa_K092300 (pTet+mRFP1), and BBa_K1455001 (pTet+amilCP).

Prologue

The selected assembly methods for our projects were based on type IIS Golden Gate assembly, restriction digestion, and Gibson assembly respectively.

Act I: Colour Parts

“I feel the beauty, which is almost entirely colour, very subtle, very changeable, running over my pen, as if you poured a large jug of champagne over a hairpin.” - Virginia Woolf, “The Letters of Virginia Woolf: Volume Six, 1936-1941”

In order to achieve the biosynthesis by the bacterial machinery of protein-based dyes we designed the following parts:

Part name	Registry code	Part type	Description
Hydrophobic tag	BBa_K2906007	Basic	Adaptation from Y3P2-GFPuv consistent of YYYPP to allow interaction with the hair cuticle (expected to embed in the hydrophobic outer β-layer of hair).
pTet+sfGFP (untagged sfGFP, Colour alone)	BBa_K2906000	Composite- improved	Untagged sfGFP CDS regulated by the TetR/A promoter.
pTet+hydrophobic tag + N-terminal secretion signal (OmpA) + sfGFP (N-terminal construct)	BBa_K2906001	Composite	Hydrophobic tagged attached to sfGFP. Expected secretion due to the addition of an N-terminal secretion signal from OmpA. Expected to become trafficked through a type II secretion system and become cleaved.
pTet+hydrophobic tag + sfGFP + C-terminal secretion signal (HlyA) (C-terminal construct)	BBa_K2906002	Composite	Hydrophobic tagged attached to sfGFP. Expected secretion due to the addition of a C-terminal secretion signal, HlyA. Expected trafficking through type I secretion system, remains attached after translocation..
pTet+hydrophobic tag + N-terminal secretion signal (OmpA) + mRFP1 (N-terminal construct)	BBa_K2906005	Composite	Hydrophobic tagged attached to mRFP1. Expected secretion due to the addition of an N-terminal secretion signal from OmpA. Expected to become trafficked through a type II secretion system and become cleaved.
pTet+hydrophobic tag + mRFP1 + C-terminal secretion signal (HlyA) (C-terminal construct)	BBa_K2906006	Composite	Hydrophobic tagged attached to mRFP1. Expected secretion due to the addition of a C-terminal secretion signal, HlyA. Expected trafficking through type I secretion system, remains attached after translocation..
pTet+hydrophobic tag + N-terminal secretion signal (OmpA) + AmilCP (N-terminal construct)	BBa_K2906003	Composite	Hydrophobic tagged attached to AmilCP. Expected secretion due to the addition of an N-terminal secretion signal from OmpA. Expected to become trafficked through a type II secretion system and become cleaved.secretion system and become cleaved.
pTet+hydrophobic tag + AmilCP+ C-terminal secretion signal (HlyA) (C-terminal construct)	BBa_K2906004	Composite	Hydrophobic tagged attached to AmilCP. Expected secretion due to the addition of a C-terminal secretion signal, HlyA. Expected trafficking through type I secretion system, remains attached after translocation..

Additionally, we used and characterised previously registered iGEM parts, as depicted in the table below:

Part name	Registry code	Part type	Description	BioBrick
pTet+mRFP1 (untagged mRFP1, Colour alone)	BBa_K2906000	Composite- previously registered iGEM part	Untagged mRFP1 CDS regulated by the TetR/A promoter.
pTet+AmilCP (untagged AmilCP, Colour alone)	BBa_K1455001	Composite- previously registered iGEM part	Untagged AmilCP CDS regulated by the TetR/A promoter.

Act II: Fragrance Parts

“Shower your head with flowers” - Suzy Kassem, “Mother is Water”, in Rise Up and Salute the Sun

The following parts were designed in order to ultimately produce vainillin intermediates this would ultimately allow our engineered bacteria to produce a pleasant scent in situ when attached to hair.

Part name	Registry code	Part type	Description
His-TEV-Sam8	BBa_K2906011	Basic	sam8 from Saccharothrix espanaensis codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag and a TEV cleavage site
T7 promoter- RBS- Sam8- T7 terminator	BBa_K2906012	Composite	sam8 from Saccharothrix espanaensis codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag and a TEV cleavage site. Under the control of a T7 promoter, lac operator, RBS and T7 terminator that are fused after cloning into pETM-11 plasmid.
His-TEV-Sam5	BBa_K2906013	Basic	sam5 from Saccharothrix espanaensis codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag and a TEV cleavage site.
T7 promoter- RBS- Sam5- T7 terminator	BBa_K2906014	Composite	sam5 from Saccharothrix espanaensis codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag and a TEV cleavage site. Under the control of a T7 promoter, lac operator, RBS and T7 terminator that are fused after cloning into pETM-11 plasmid.
His-COMT	BBa_K2906015	Basic	comt from Arabidopsis thaliana codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag.
T7 promoter- RBS- COMT- T7 terminator	BBa_K2906016	Composite	comt from Arabidopsis thaliana codon optimised for expression in E. coli (GeneArt GeneOptimiser) and fused to a His-tag. Under the control of a T7 promoter, lac operator, RBS and T7 terminator that are fused after cloning into pETDuet-1 plasmid.
Fcs	BBa_K2906021	Basic	fcs from Pseudomonas putida KT2440 codon optimised for expression in E. coli.
Ech	BBa_K2906023	Basic	ech from Pseudomonas putida KT2440 codon optimised (GeneArt GeneOptimiser) for expression in E. coli.
T7 promoter - lac operator	BBa_K2906019	Basic	A strong T7 promoter fused to a lac operator found on pETM-11 and pETDue-1 plasmids.
RBS	BBa_K2906017	Basic	A strong RBS from bacteriophage T7, found in pETM-11 and pETDuet-1 plasmids.

We additionally also produced limonene. However, the used DNA is not iGEM standardised and therefore we were unable to submit it as a Biobrick (to learn more about our limonene production, please go to our fragrance page).

Act III: Repair Parts

“If it ain’t broke…” - a popular saying that does not apply here

We aimed at the production of a small peptide able to interact with the keratin of hair in the cortex. However, this peptide only has 11 amino acids and therefore we feared detection could be difficult. For that reason, we produced our parts both with the small protein as well as with a histidine tag for detection purposes only. The designed parts are shown below:

Part name	Registry code	Part type	Description
PepG	BBa_K2906100	Basic	A cysteine rich peptide is produced from this gene. This peptide has been shown to not only straighten hair, but also repair it. It is though that this is achieved by this peptide entering the cortex of the hair and reforming disulfide bonds.
PepG + His tag	BBa_K2906101	Composite	The pepG peptide that has been reported to straighten and strengthen hair, but with the addition of a his-tag to enable purification and detection.
PepG + His tag + SecB dependent N-terminal secretion tag (OmpA)	BBa_K2906102	Composite	The pepG peptide that has been reported to straighten and strengthen hair, but with the addition of a his-tag to enable purification and detection as well as an ompA tag to enable secretion.The ompA tag gas been chosen as it has been shown to be cleaved upon secretion, given the size of the peptide, removal of the tag will enable function.
PepG + SecB dependent N-terminal secretion tag (OmpA)	BBa_K2906103	Composite	This time the pepG peptide has been fused the SecB dependent N-terminal secretion tag (ompA) only.

Team:Manchester/Part Collection