www.company.com
GB2.0
GOLDENBRAID 2.0: A comprehensive DNA assembly framework for Plant Synthetic
Biology and Molecular Farming
Plant Genomics and Biotechnology GroupIBMCP (CSIC-UPV), Valencia
www.company.com
GB2.0
www.gbcloning.org
Plant Synthetic Biology
Multigenic traits
Plant Genetic Engineering
Single gene traits
MOLECULAR FARMING
The transit from Biotechnology to Synthetic Biology
www.company.comwww.gbcloning.org
GB2.0
The benefits of Modular Design
STA
ND
AR
DIZ
AT
ION
HIGH-THROUGHPUT
FLEXIBILITY
INTERCHANGEABILITY
www.company.comwww.gbcloning.org
GB2.0
GB2.0
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5 UTR LINK 3 UTR
01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)
SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)
CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)
NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)
NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)
OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)
PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)
TL1 TL2 TL4TL3
16 (CT)
17
01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)
16B (3’FS) 17-21 (TER)
GTAG TCTC
amiRNA
01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)
01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)
hpRNA
tasiRNA
TGAC
03
02 (OP)
OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP)
02468
1012141618
PNos 0 μM 5 μM 10 μM P35s
No
rma
lize
d F
Lu
c/R
Lu
c
Grammar
Database
Functionally-tested genetic modules
The GoldenBraid 2.0 Framework
Software tools
Interchangeability
www.company.com
GB2.0
www.gbcloning.org
Type IIS enzymeType II enzyme
nnnn cctgag ctcgag
Type IIS restriction enzymes
GoldenGate:
Engler, C., R. Gruetzner, R. Kandzia, and S. Marillonnet, PLoS One, 2009. 4(5): p. e5553.Engler, C., R. Kandzia, and S. Marillonnet, PLoS One, 2008. 3(11): p. e3647.
GoldenBraid 1.0:
Sarrion-Perdigones, A., E.E. Falconi, S.I. Zandalinas, P. Juarez, A. Fernandez-Del-Carmen, A. Granell, and D. Orzaez,. PLoS One, 2011. 6(7): p. e21622
Type IIS enzymeType II enzyme
The chemistry behind GoldenBraid
www.company.com
GB2.0
www.gbcloning.org
The chemistry behind GoldenBraid
Suffix
AATG
Prefix
GCTT
PROMOTER + UTR +ATG CDS TERMINATOR
GGAG AATG GCTT CGCT
www.company.com
GB2.0
www.gbcloning.org
d t
u
nes
ra n
d
GB 2.0: analogy with the grammar
www.company.comwww.gbcloning.org
GB2.0
understand
do
not
ityou
!
GB 2.0: analogy with the grammar
www.company.comwww.gbcloning.org
GB2.0
you do not understand it!
If you can’t compute it
GB 2.0: analogy with the grammar
www.company.com
GB2.0
www.gbcloning.org
GBparts Transcriptional Units
Genetic Modules
Multipartite
Binary,Iterative,
GB works as a double iterative loop that allows the stepwise assembly of new TUs indefinitely.
GB supports multipartite assemblies of several (up to 8) small DNAparts in a single reaction to create a transcriptional units
The GB 2.0 assembly strategy
Basic Genetic elements
www.company.com
GB2.0
www.gbcloning.org
pDGB α213 2
pDGB Ω23 21
pDGB α121 LacZ
pDGB Ω11 323
LacZ
LacZ
LacZ
α L
EV
EL
(B
saI r
eact
ion
s) Ω L
EV
EL
(Bsm
BI reactio
ns)
The 4 pDGBs give a double-loop structure (braid)
www.company.com
GB2.0
www.gbcloning.org
GB database
GB TU2
SEQUENCE
GB partGB part
GB TU1
GB Module
GB Domesticator
GB TU Assembler
GB Binary Assembler
GB part GB part
GB 2.0 software tools
www.company.comwww.gbcloning.org
GB2.0
35S Jchain TNos
35S SP TNos
35S SC TNos
CLVL
35S SP TNosCHVH
SP
SP
GoldenBraid 2.0: building a secretory IgA
www.company.comwww.gbcloning.org
GB2.0The GB2.0 workflow
35S
SC
TNos
SP
TNos
35S
SP
hSC DOMESTICATION
AGCC
GCTT
AATG
CGCT
GGAG
AATG
GCTTAGCC MULTIPARTITE ASSEMBLY
35S SC TNosSP
PARTS and SUPERPARTS
TRANSCRIPTIONAL UNIT
BINARYASSEMBLY
35S Jchain TNos
35S SC TNosSP
SP
GENETICMODULE
www.company.comwww.gbcloning.org
GB2.0
SUBJECT PREDICATE STOP
VERB ART NOUNNOUN
- English sentence
The Grammar
ADJ
Biotechnology
has a hugepotential .
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5´UTR LINK 3´UTR
01-02-03-11-12 (PROM+UTR+ATG)13
(SP) 14-15-16 (CDS) 17-21 (TER)
TL1 TL2 TL4TL3
17
TGAC
03
35S SC TNosSP
- Transcriptional Unit
ART
www.company.comwww.gbcloning.org
GB2.0
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5 UTR LINK 3 UTR
01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)
SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)
CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)
NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)
NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)
OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)
PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)
TL1 TL2 TL4TL3
16 (CT)
17
01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)
16B (3’FS) 17-21 (TER)
GTAG TCTC
amiRNA
01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)
01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)
hpRNA
tasiRNA
TGAC
03
02 (OP)
OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP)
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5 UTR LINK 3 UTR
01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)
SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)
CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)
NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)
NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)
OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)
PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)
TL1 TL2 TL4TL3
16 (CT)
17
01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)
16B (3’FS) 17-21 (TER)
GTAG TCTC
amiRNA
01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)
01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)
hpRNA
tasiRNA
TGAC
03
02 (OP)
OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP) SC
AGCC GCTT
Domestication of the Secretory Component
www.company.comwww.gbcloning.org
GB2.0
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5 UTR LINK 3 UTR
01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)
SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)
CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)
NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)
NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)
OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)
PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)
TL1 TL2 TL4TL3
16 (CT)
17
01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)
16B (3’FS) 17-21 (TER)
GTAG TCTC
amiRNA
01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)
01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)
hpRNA
tasiRNA
TGAC
03
02 (OP)
OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP)
TTCGAGCC GCAGGGAG TCCC TACT CCAT AATG GCTT GGTA CGCT
1311 12 14 15 16 2101 02
5’ NT TRANSCRIBED REGION 3’ NT
5 UTR LINK 3 UTR
01-02-03-11-12 (PROM+UTR+ATG) 13-14-15-16 (CDS) BASIC17-21 (TER)
SECRETED01-02-03-11-12 (PROM+UTR+ATG) 13 (SP) 14-15-16 (CDS) 17-21 (TER)
CT-FUSION01-02-03-11-12 (PROM+UTR+ATG) 16 (CT)13-14-15 (CDS) 17-21 (TER)
NT-FUSION13-14-15-16 (CDS)12 (NT)01-02-03-11 (PROM+UTR) 17-21 (TER)
NT & CT FUSION12 (NT) 13-14 (CDS)01-02-03-11 (PROM+UTR) 17-21 (TER)
OP PROM-B13-14-15 -16(CDS)03-11-12 (MinPROM)01 (PROM) 17-21 (TER)
PROT INTERACT13-14-15-16 (CDS)01-02-03-11-12B (INTERACTION ADAPTOR) 17-21 (TER)
TL1 TL2 TL4TL3
16 (CT)
17
01-02-03-11 (PROM+UTR) 12-13B (5’FS)14B-15B (Target)
16B (3’FS) 17-21 (TER)
GTAG TCTC
amiRNA
01-02-03-11 (PROM+UTR) 12-13(GOI) 16 (IOG)14-15 (INT) 17-21 (TER)
01-02-03-11-C (PROM+UTR+mir173) 12-13-14-15-16 (fGOI) 17-21 (TER)
hpRNA
tasiRNA
TGAC
03
02 (OP)
OP PROM-A13-14-15 -16(CDS)03-11-12 (MinPROM)01 (OP) 17-21 (TER)01-02 (OP) 35S SC TNosSP
Multipartite Assembly of the TU encoding the SC
GB DB GB DB GB DB
DOMESTICATED
www.company.comwww.gbcloning.org
GB2.0
pDGB_Ω23 2LacZ
TU SC
1 3
TU JC
3 2
1
3 2
Binary Assembly of the TUs encoding the SC and the JC
www.company.comwww.gbcloning.org
GB2.0
pDGB_α1
21 3
TU HC TU LC
31
3 2
1
LacZ
TU JCTU SC
3
Binary Assembly of the preassembled genetic modules
www.company.comwww.gbcloning.org
GB2.0
Secretory IgA construction
4 TUs: 13kb
www.company.comwww.gbcloning.org
GB2.0
The major benefit of GB2.0 framework: interchangeability
Every story has an end, but with GB every ending is just a new beginning.
www.company.comwww.gbcloning.org
GB2.0
GB2.0 is a convenient framework for Plant Synthetic Biology, including Molecular Farming and Metabolic
Engineering
To learn more: www.ibmcp.upv.es/FGBwww.gbcloning.org
Conclusions
Hands-on course on multiple gene assembly technologies for metabolic engineering and fruit biotechnology
Valencia (Spain), September 9th to 13th, 2013
Organized by COST Actions FA1006 (PlantEngine) and FA1106 (QualityFruit)
http://www.plantengine.eu/node/156; http://qualityfruit.inp-toulouse.fr/en/training/dna-assembly.html
www.company.com
THANK YOU!
IBMCPAlejandro Sarrión
Javier FormentPaloma JuárezEstefanía Huet
Asun FernándezJose Manuel Julve
Diego Orzáez
Fruit genomics and Biotechnology Group.
www.ibmcp.upv.es/FGB
COMAVPeio Ziarsolo
Jose Blanca
Top Related