Collection

Fig.1 Results of yeast toolkit plasmids enzyme-digested verification

Part:BBa_K4012001

pTEF1

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pTEF1(709bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012002

pGAL1

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pGAL1(542bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012003

pPGK1

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments Type-4-tPGK1(238bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012004

pPOP6

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pPOP6(709bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012005

pRET2

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pRET2(709bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012006

pTDH3

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pTDH3(689bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012007

pTEF2

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pTEF2(709bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012008

pAOX1

It is a promoter from S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pAOX1(943bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012019

tTDH1

It is a terminator in S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tTDH1(237bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012020

tSSA1

It is a terminator in S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tSSA1(238bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012021

tENO2

It is a terminator in S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tENO2(238bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012022

tADH1

It is a terminator in S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tADH1(238bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012023

tCYC1

It is a terminator in S. cerevisiae genome. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments tCYC1(259bp) matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

Part:BBa_K4012010

GbCHS

The construction results of coding sequence GbCHS are shown by the Fig.6:

Fig.6 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GbCHS

GbCHS is sequence in Ginkgo biloba that perform Chalcone synthase.The construction schematic of GbCHS sequence is demonstrated in Fig.6. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.6 B The band length 2529bp matched with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012011

AaDFR( Arabidopsis thaliana dihydroflavonol 4-reductase)

The construction results of coding sequence AaDFR are shown by Fig.11:

Fig.11 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence AaDFR

AaDFR is sequence in Anthurium andraeanum that perform dihydroflavonol 4-reductase,also called Tetraketide alpha-pyrone reductase 1.The construction schematic of AaDFR sequence is demonstrated in Fig.11. The initiation of the AaDFR sequence is done by promoter pTDH3, with termination done by tENO1. The sequence ConL3 and ConR4 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.11 B The band length 2377bp matched with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012012

CrCPR(Catharanthus roseus NADPH--cytochrome P450 reductase sequence)

The construction results of coding sequence CrCPR are shown by Fig.8:

Fig.8 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CrCPR

CrCPR is sequence in Catharanthus roseus that perform NADPH--cytochrome P450 reductase. The construction schematic of CrCPR sequence is demonstrated in Fig.8. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.8 B The band length 3327bp matched with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012013

CsF3H (Camellia sinensis flavanone 3-hydroxylase)

The construction results of coding sequence CsF3H are shown by Fig.9:

Fig.9 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CsF3H

CsF3H is sequence in Camellia sinensis that perform flavanone 3-hydroxylase or called Flavonol synthase. The construction schematic of CsF3H sequence is demonstrated in Fig.9. The initiation of the CsF3H sequence is done by promoter pGAL7, with termination done by tENO2. The sequence ConL1 and ConR2 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.9 B The band length 2755bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012014

DuLAR(Desmodium uncinatum Leucoanthocyanidin reductase)

The construction results of coding sequence DuLAR are shown by Fig.12:

Fig.12 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence DuLAR

DuLAR is sequence in Desmodium uncinatum that perform leucoanthocyanidin reductase. The construction schematic of DuLAR sequence is demonstrated in Fig.12. The initiation of the DuLAR sequence is done by promoter pPOP6, with termination done by tSSA1. The sequence ConL4 and ConRE are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.12 B The band length 2502bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012015

GhF3'H

The construction results of coding sequence GnF3’H are shown by Fig.10:

Fig.10 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GhF3’H

GhF3’H is sequence in Gerbera hybrid cultivar that perform flavonoid 3'-monooxygenase. The construction schematic of GhF3’H sequence is demonstrated in Fig.10. The initiation of the GnF3’H sequence is done by promoter pPGK1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.10 B The band length 2892bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012016

MsCHI

The construction results of coding sequence MsCHI are shown by Fig.7:

Fig.7 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence MsCHI

MsCHI is sequence in Medicago sativa that perform Medicago sativa chalcone isomerase. The construction schematic of MsCHI sequence is demonstrated in Fig.7. The initiation of the MsCHI sequence is done by promoter pAOX1, with termination done by tTDH1. The sequence ConL3 and ConRE are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.7 B The band length 2021bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012017

Pc4CL

The construction results of coding sequence Pc4CL are shown by Fig.5:

Fig.5 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence Pc4CL

Pc4CL is sequence in Petroselinum crispum that perform Medicago sativa chalcone isomerase. The construction schematic of Pc4CL sequence is demonstrated in Fig.5. The initiation of the Pc4CL sequence is done by promoter pPGK1, with termination done by tCYC1. The sequence ConL1 and ConR2 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.5 B The band length 3004 bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012018

RtTAL

The construction results of coding sequence RtTAL are shown by Fig.4:

Fig.4 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence RtTAL

RtTAL is sequence in Rhodotorula glutinis that perform tyrosine ammonia lyase. The construction schematic of RtTAL sequence is demonstrated in Fig.4. The initiation of the RtTAL sequence is done by promoter pRET2, with termination done by tSSA1. The sequence ConLS and ConR1 are connector sequences used within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are primers used to enact selection through colonies PCR, results are shown in Fig.4 B. The band length 3432bp match with the expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

Part:BBa_K4012029

RtTAL-Pc4CL-GbCHS-MsCHI level2

It is a composite part that perform the synthesis of naringenin.

Part:BBa_K4012030

CrCPR-CsF3H-GhF3'H-AaDFR-DuLAR level2

It is a composite part that perform the synthesis of catechin.

The results of junctions PCR are shown in Fig.14.

Fig.14 A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

The results in Fig.14 show clear bands with length that matched with the expectations. The subsequent sequence analysis shows no significant mutations or deletions, confirming the success of construction of two metabolism pathways.

Team:AISSU Union/Collection