Team:Athens/Biobricks

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BIOBRICKS

Overview

The listed parts were designed by iGEM Athens 2020 during the project MORPHÆ. In this project Flavobacteriia were used to produce a non-cellular structurally coloured biomaterial which would require the secretion of a biomolecule that they do not normally secrete. Our hypothesis is that the formed matrix will have a structure similar to that of the biofilm and thus, it will provide the material with macroscopically the same colouration properties as the biofilm.

Our initial idea was to use bacterial cellulose, as an appropriate biomaterial, because of its unique properties, robustness and biodegradability [1]. The genes responsible for its production were selected from the bcs operon of Komagataeibacter xylinus (GenBank Acc. No. X54676.1), the most efficient bacterial cellulose producer, which consists of four genes, bcsA, bcsB, bcsC and bcsD. The bcsABCD operon encodes membrane associated proteins that allow BC fibres to span through the membrane.

We wholeheartedly hope all of the designed parts will make it easier for future teams that work with the particular species and give them higher manipulation capabilities and accuracy. This applies for teams which aim to produce cellulose with genetic manipulation of the particular or any other bacteria as well.

Part Name	Short Description	Part Type	Length (bp)
BBa_K3520031	bcsA-Bacterial Cellulose Synthase A	Coding	2172
BBa_K3520002	bcsB-Regulatory Subunit of Bacterial Cellulose operon	Coding	2483
BBa_K3520003	bcsC- part of Bacterial Cellulose operon	Coding	3909
BBa_K3520004	bcsD-part of Bacterial Cellulose operon	Coding	478
BBa_K3520005	cmcax: gene for Bacterial Cellulose upregulation	Coding	1029
BBa_K3520006	ccpAx: gene for Bacterial Cellulose upregulation	Coding	1062
BBa_K3520007	ompA promoter for Flavobacteriia	Regulatory element	90
BBa_K3520008	Flavobacteriia RBS	RBS	5
BBa_K3520009	pHimarEm1	Plasmid backbone	6656

Part Name	Short Description	Part Type	Length (bp)
BBa_K3520013	Flavobacteriia compatible BcsA transcriptional unit	Transcriptional Unit	2370
BBa_K3520014	Flavobacteriia compatible BcsB transcriptional unit	Transcriptional Unit	2625
BBa_K3520015	Flavobacteriia compatible BcsC transcriptional unit	Transcriptional Unit	4106
BBa_K3520016	Flavobacteriia compatible BcsD transcriptional unit	Transcriptional Unit	665
BBa_K3520019	pMORPHÆ:recombinant pHimarEm1with the insert of BcsABCD transcriptional units	Plasmid	16450
BBa_K3520021	Flavobacteriia compatible cmcAx transcriptional unit	Transcriptional Unit	2369
BBa_K3520022	Flavobacteriia compatible cpAx transcriptional unit	Transcriptional Unit	1237

In order for each basic part to form a transcriptional unit with a promoter, RBS and terminator they were flanked with the appropriate suffixes and prefixes.

The assembly method we are planning on following is the Type IIS assembly standard, but the prefixes and suffixes will also contain the RFC10 respectively. BioBricks design is the same for every Basic Part with the only difference being a four nucleotide sequence, in order for the assembly to happen with the desired synteny. Once Level 0 → 1 is completed, the promoter and terminator were additionally flanked with the SapI restriction site and the three nucleotide sequence appropriate for the Type IIS Level 1→ 2 assembly standard. Once each transcriptional unit is formed, they are digested with SapI and ligated according to the following standard.

Special sequence for basic parts (Level 0)

Prefix	Part	Suffix
5' GGAG 3' CCTC	Promoter	TACT 3' ATGA 5'
5' TACT 3' ATGA	RBS	AATG 3' TTAC 5'
5' AATG 3' TTAC	CDS	GCTT 3' CGAA 5'
5' GCTT 3' CGAA	Terminator	CGCT 3' GCGA 5'

Special sequence for composite Parts (Level 1)

Prefix	Part	Suffix
5' GGAG 3' CCTC	Promoter	TACT 3' ATGA 5'
5' TACT 3' ATGA	RBS	AATG 3' TTAC 5'
5' AATG 3' TTAC	CDS	GCTT 3' CGAA 5'
5' GCTT 3' CGAA	Terminator	CGCT 3' GCGA 5'

igemathens2020@gmail.com