To reach our goals with oPHAelia, our team designed 28 basic parts and 39 composite parts. We tested 9 basic parts, which we then combined to create 17 composite parts. On this page you can find constructs we worked on, as well as other components we utilized such as primers we designed and our cloning vectors. For better comprehension of the GoldenBraid cloning terms on this page we encourage you to first read the section “Cloning method” on our Engineering page.

All of our basic parts were domesticated to remove any internal restriction sites for the BsmBI and BsaI Type IIS restriction enzymes and 4nt-overhangs were added compatible with the grammar of GoldenBraid 2.0. The parts we cloned are listed below.

Basic Parts
Part name	Registry Code	Part Type	Golden Braid Overhangs	Length (bp)
PFR1	BBa_K4624000	Regulatory	A1-B2	66
PfadBA	BBa_K4624625	Regulatory	A1-B2	81
PLR	BBa_K4624602	Regulatory	A1-B2	72
PAR	BBa_K4624601	Regulatory	A1-B2	77
Na.SP	BBa_K4624400	Signalling	B3	63
fapR	BBa_K4624003	Coding	B3-B5 and B4-B5	567
laccase	BBa_K4624004	Coding	B4 and B4-B5	1500
fadR	BBa_K4624005	Coding	B3-B5 and B4-B5	720
Linker peptide	BBa_K4624006	Protein domain	-	33

Just like our basic parts, our composite parts were designed in accordance with the Golden Braid 2.0 grammar, an assembly system that operates with a double iterative loop. Thanks to this characteristic, our basic parts (level 0) with the correct compatible 3’ and 5’ overhangs can be combined to create a transcriptional unit into a level alpha plasmid. Subsequently, two level alpha plasmids, each carrying a transcriptional unit, can be combined to generate a level omega plasmid, where the two transcriptional units are combined. Below we have listed all of the level 1 (alpha) and level 2 (omega) composite parts we tested during this iGEM season.

Level 1 Composite parts
Transcription Unit	Registry Code	GB cloning vector	Length (bp)
PFR1-syfp2-rrnB T1/T7TE	BBa_K4624010	α1	959
AraC/PBAD-syfp2-rrnB T1/T7TE	BBa_K4624618	α1	2227
AraC/PBAD-fapR-rrnB T1/T7TE	BBa_K4624619	α2	2071
AraC/PBAD-fapR-syfp2-rrnB T1/T7TE	BBa_K4624620	α1	2830
PJ23118-syfp2-rrnB T1/T7TE	BBa_K4624621	α1	930
PfadBA-syfp2-rrnB T1/T7TE	BBa_K4624622	α1	963
PLR-syfp2-rrnB T1/T7TE	BBa_K4624624	α1	954
PAR-syfp2-rrnB T1/T7TE	BBa_K4624623	α1	959
AraC/PBAD-Na.SP-laccase-rrnB T1/T7TE	BBa_K4624627	α1	3073
AraC/PBAD-NSP4-laccase-rrnB T1/T7TE	BBa_K4624628	α1	3070
AraC/PBAD-PelB-laccase-rrnB T1/T7TE	BBa_K4624629	α1	3076
AraC/PBAD-MalE-laccase-rrnB T1/T7TE	BBa_K4624630	α1	3088
AraC/PBAD-Na.SP-laccase-syfp2-rrnB T1/T7TE	BBa_K4624631	α1	3832
AraC/PBAD-NSP4-laccase-syfp2-rrnB T1/T7TE	BBa_K4624632	α1	3829
AraC/PBAD-PelB-laccase-syfp2-rrnB T1/T7TE	BBa_K4624633	α1	3835
AraC/PBAD-MalE-laccase-syfp2-rrnB T1/T7TE	BBa_K4624634	α1	3847

Level 2 Composite parts
Transcription Unit	Part Code	GB cloning vector	Length (bp)
PFR1-syfp2-rrnB T1/T7TE + AraC/PBAD-fapR-rrnB T1/T7TE	BBa_K4624626	ω1	3034

Level 0 Composite parts
Part name	Registry Code	GB cloning vector	Length (bp)
Linker-syfp2	BBa_K4624007	pUPD2	756

Part code	Name	Description	Origin
BBa_K3505007	pUPD2	Level 0 vector for GoldenBraid assembly	iGEM Thessaly 2020
BBa_K4213058	pDGB3alpha1 (α1)	Level 0 vector for GoldenBraid assembly	iGEM Thessaly 2022
BBa_K4213059	pDGB3alpha2 (α2)	Level 1 vector for GoldenBraid assembly	iGEM Thessaly 2022
BBa_K4213060	pDGB3omega1 (ω1)	Level 2 vector for GoldenBraid assembly	iGEM Thessaly 2022

Due to the nature of Golden braid, certain 3’ and 5’ overhangs can be used only for one position in the transcription unit. This meant that, in our case, for certain parts such as the protein coding sequences, we had to add the different overhangs with PCR. Below is the list with the primers we designed.

Part code	Description	Length (bp)
BBa_K4624603	fadR B3+B4+(B5) Forward	37
BBa_K4624604	fadR B3+B4 Reverse	41
BBa_K4624605	fadR B3+B4+(B5) Reverse	39
BBa_K4624606	fapR B3+B4+(B5) Forward	41
BBa_K4624607	fapR B3+B4 Reverse	45
BBa_K4624608	fapR B3+B4+B5 Reverse	45
BBa_K4624609	accCD B4+(B5) Forward	43
BBa_K4624610	accCD B4 Reverse	41
BBa_K4624611	accCD B4+B5 Reverse	42
BBa_K4624612	laccase B4+(B5) Forward	37
BBa_K4624613	laccase B4 Reverse	40
BBa_K4624614	laccase B4+B5 Reverse	41
BBa_K4624615	lacI B3+B4+(B5) Forward	40
BBa_K4624616	lacI B3+B4 Reverse	41
BBa_K4624617	lacI B3+B4+(B5) Reverse	41

We aspire that our input into enriching the iGEM registry will provide upcoming teams with valuable information, resources, and inspiration to create.