Parts | WWU_Muenster

Summary

Listed below are the crucial parts that we obtained during our project. Because they are based on an established vector set, we described changes in the following table omitting features of the plasmid backbones that are already described in the iGEM registry. We submitted 30 new parts in total that are part of two new collections.

Shuttle vectors

We particularly promote our shuttle vectors (the trans-Yeast collection) that we developed through our partnership with the iGEM Team TU Dresden (Table 1). They are designed to make the model organism Saccharomyces cerevisiae more accessible to future iGEM teams, so the new features of these vectors are focused particularly on simple handling and a high degree of flexibility.

Table 1: List of the designed shuttle vectors

Name	Type	Description	length (bp)
BBa_K4188001	Plasmid_Backbone	Designed shuttle vector for S. cerevisiae transformations containing the 2µ ori and the chromoprotein aeBlue expression cassette. The latter is flanked by BsmBI restriction sites for easy introduction of a selection marker. Moreover, the shuttle vector includes the chromoprotein mRFP1 expression cassette between SapI restriction sites for insertion of a multi-transcription unit expressible in yeast.	6175
BBa_K4188002	Plasmid	Ready to use version of the BBa_K4188001 part with a histidine synthase (HIS3) cassette.	6303
BBa_K4188003	Plasmid	Ready to use version of the BBa_K4188001 part with a leucine synthase (LEU2) cassette.	7110
BBa_K4188004	Plasmid	Ready to use version of the BBa_K4188001 part with a tryptophane synthase (TRP1) cassette.	6818
BBa_K4188005	Plasmid	Ready to use version of the BBa_K4188001 part with an uracil synthase (URA3) cassette.	6252

Basic parts for the cytosolic expression

In Table 2, all submitted basic parts are listed required for the up regulation of the mevalonate pathway in the cytosol of S. cerevisiae. All basic parts were submitted cloned in the pSB1C00.

Table 2: List of basicparts submitted for cytosolic expression.

Part	Type	Description	length (bp)
BBa_K4188020	Coding Sequence	ApL3H coding sequence for the catalysis from α-pinene to verbenone.	1584
BBa_K4188022	Coding Sequence	GgmFPS144 coding sequence from chicken as a replacement for the ERG20 which produces more GPP (geranyl pyrophosphate) than farnesyl pyrophosphate (FPP).	1104
BBa_K4188023	Coding Sequence	AgtCS coding sequence for the synthesis from GPP to camphene. Truncated version without chloroplast tag.	1707
BBa_K4188024	Coding Sequence	SltNPPS1 coding sequence for the synthesis of neryl pyrophosphate (NPP). Truncated version without chloroplast tag.	783
BBa_K4188025	Coding Sequence	AgtGPPS2 coding sequence for the upregulation of the GPP content in the cytosol.	891
BBa_K4188026	Coding Sequence	ScERG13 coding sequence which catalyzes the reaction of acetoacetyl-CoA to HMG-CoA.	1476
BBa_K4188027	Coding Sequence	SctHMGR coding sequence to remove the limiting step in the mevalonate pathway. Truncated version without repression side.	1509
BBa_K4188028	Coding Sequence	TvtGTPS1 coding sequence catalysis the reaction from GPP to γ-terpinene. Truncated version without chloroplast tag.	1629
BBa_K4188029	Coding Sequence	TvSDR1 coding sequence. As part of thymol biosynthesis, it catalysis the reaction of the dienol intermediate to the allylic ketone intermediate, which undergoes keto-enol tautomerisms to become thymol.	795
BBa_K4188030	Coding Sequence	TvCYP71D179 coding sequence hydroxylates γ-terpinene at the third C atom to an dienol intermediate.	1491
BBa_K4188031	Coding Sequence	PptAPS coding sequence for the catalysis from GPP/NPP to α-pinene. Truncated version without chloroplast tag.	1746
BBa_K4188034	Coding Sequence	afraGFP coding sequence for cytosolic expression.	681

Basic parts for the peroxisomal expression

Table 3 lists all parts required to integrate the mevalonate pathway into the peroxisome of S. cerevisiae. All parts to be found here contain the PTS1 tag which ensures the integration in the peroxisome. All basic parts were submitted cloned in the pSB1C00.

Table 3: List of submitted basic parts that have been tagged with a peroxisome tag.

Part	Type	Description	length (bp)
BBa_K4188006	Coding Sequence	afraGFP coding sequence fused to the PTS1 tag for the peroxisomal expression.	690
BBa_K4188007	Coding Sequence	ScERG8 coding sequence fused to the peroxisomal tag PTS1. Catalyzes the reaction of mevalonate-5-phosphates to mevalonate-5-pyrophosphates.	1365
BBa_K4188008	Coding Sequence	ScERG19 coding sequence fused to the peroxisomal tag PTS1.	1200
BBa_K4188009	Coding Sequence	ScERG13 coding sequence fused to the peroxisomal tag PTS1. Catalyzes the reaction of acetoacetyl-CoA to HMG-CoA.	1485
BBa_K4188010	Coding Sequence	ScERG10 coding sequence fused to the peroxisomal tag PTS1. Catalyzes the reaction of acetyl-CoA to acetoacetyl-CoA.	1206
BBa_K4188011	Coding Sequence	ScIDI1 coding sequence fused to the peroxisomal tag PTS1. Catalyzes the reaction of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and reverse for an equilibrium of these two metabolites.	876
BBa_K4188012	Coding Sequence	ScERG12 coding sequence fused to the peroxisomal tag PTS1. Catalyzes the reaction of mevalonate to mevalonate-5-phosphate.	1341
BBa_K4188013	Coding Sequence	SltNPPS1 coding sequence fused to the peroxisomal tag PTS1. Catalysis the reaction IPP and DMAPP to NPP. Truncated version without chloroplast tag.	792
BBa_K4188014	Coding Sequence	AgtGPPS2 coding sequence fused to the peroxisomal tag PTS1. Synthesizes the required amounts of GPP.	900
BBa_K4188015	Coding Sequence	SctHMGR coding sequence to remove the limiting step in the mevalonate pathway. Truncated version without repression side and fused to the PTS1 tag for peroxisomal expression.	1518
BBa_K4188016	Coding Sequence	GgmFPS144 coding sequence which produces more GPP than FPP. Fused to the PTS1 tag for peroxisomal expression.	1113
BBa_K4188019	Coding Sequence	TvtGTPS1 coding sequence fused to the peroxisomal tag PTS1. Catalysis the reaction from GPP to γ-terpinene. Truncated version without chloroplast tag.	1638
BBa_K4188032	Coding Sequence	PptAPS coding sequence for the catalysis from GPP/ NPP to α-pinene. Truncated version without chloroplast tag and fused to the PTS1 tag for peroxisomal expression.	1755

Used basic parts

Finally, we listed all basic Parts from the iGEM Registry in Table 4 that were used for cloning expression cassettes in MonChassis.

Table 4: List of used Basic parts from the iGEM registry

Part	Type	Description	length (bp)
BBa_K1430000	Coding Sequence	The 2µ-ori for replication in S. cerevisiae.	1343
BBa_K864401	Coding Sequence	The aeBlue coding sequence from the Uppsala chromoprotein collection.	699
BBa_J23110	Promotor	The constitutive promoter J23110 from the Anderson collection.	35
BBa_B0015	Terminator	The double Terminator	129
pSB1C00	Plasmid_Backbone	iGEM MoClo Lv0 Backbone	2053
pSB1K01	Plasmid_Backbone	iGEM MoClo Lv1 Backbone	2195
pSB1K02	Plasmid_Backbone	iGEM MoClo Lv1 Backbone	2195
pSB1K03	Plasmid_Backbone	iGEM MoClo Lv1 Backbone	2195
pSB1K04	Plasmid_Backbone	iGEM MoClo Lv1 Backbone	2195
pSB3C01	Plasmid_Backbone	iGEM MoClo Lv2 Backbone	2729
pSB3C02	Plasmid_Backbone	iGEM MoClo Lv2 Backbone	2729
pSB3C03	Plasmid_Backbone	iGEM MoClo Lv2 Backbone	2729