Transcript The Agrozobium™ project

A new selectable marker gene
The GUSPlusTM project
A new GUS gene,
Available under BIOSTM licensing,
Pioneering use of the BioForgeTM concept
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Why do we want extra-cellular GUS?
• Superiority and familiarity of GUS as a reporter
protein
• Assays with E.coli GUS are almost always destructive
• Secreted protein should allow in vivo assays
• New biological possibilities with externally applied
glucuronide-conjugated biomolecules
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GUSPlusTM is a more sensitive reporter than
E.coli GUS
Rice anthers and style
showing GUSPlusTM
expression
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Properties of GUSPlusTM
√ Almost identical in size to E.coli GUS (602 vs 603aa)
√ 47% amino acid identity with E.coli GUS
√ Secreted in Staphylococcus spp. and E.coli, no signal
peptide needed
√ One cysteine vs nine in E. coli GUS
√ Excellent stability and catalytic properties
X AT-rich coding gene, giving lower expression in
heterologous systems
Synthetic GUSPlusTM gene construction
• codon usage optimised for plants
• eliminated unwanted signals: polyA, splice sites, unwanted
restriction sites
• minimised potential secondary structures
• introduced useful restriction sites
• GusPlus gene rebuilt from oligos
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GUSPlusTM has lower Km, similar Vmax to E.coli GUS
1/v (nmol-1·mg·min)
0.04
0.03
0.02
BGUS
-1/Km
-20
1/Vmax
0.01
-10
BGUS
EGUS
0
10
Km
Vmax
EGUS
40 mM
120 mM
80
80
-1
nmol.mg min-1
20
1/[pNPG] (mM -1)
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GUSPlusTM has enhanced activity, and
improved thermal stability
60°C
Relative activity (%)
100
80
60
40
GUSPlus
E.coli GUS
20
0
0
20
40
60
80
Temperature (°C)
0
10
20
30
Time (min)
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GUSPlusTM and E.coli GUS have similar,
broad pH optima
Activity (nmol·mg -1·min-1)
100
80
60
40
20
GUSPlus
E. coli GUS
0
5
6
7
8
9
pH
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GUSPlusTM has improved chemical resistances
Relative activity (%)
120
100
GUSPlus
E. coli GUS
Assayed with
0.25mM pNPGlcA
80
60
40
20
0
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GUSPlusTM with signal peptide is secreted
B
GUSPlus
ExtSP-GUSPlus
(+ Extensin signal peptide)
GRP-SP-GUSPlus
(+ GRP signal peptide)
Sections of rice root stained yellow-green by activity
of GUSPlusTM using ELF-97-GlcA substrate.
• GRP signal peptide directs GUSPlusTM to cell wall
apoplastic space more efficiently than extensin
signal peptide
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New possibilities with a secreted GUS
• Non-destructive, high sensitivity in vivo staining for
qualitative gene expression analysis
• Treatment of GUSPlus-secreting cells with
apoplastically-transportable/diffusible glucuronideconjugated effector molecules
• Use as screenable marker gene
• Use as selectable marker gene
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How will this technology be available for use by
the agricultural R&D community?
• Traditional intellectual property licenses contain
covenants under which the licensee must agree to:
• Royalties and/or milestone payments
• Exclusive or non-exclusive licence, with various restrictions
on field of use
• (often) Grantback of improvements to licensor
• (often) Assistance to licensor in maintaining patent monopoly
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GusPlus is available for use
under the conditions of a BIOS license
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Traditional intellectual property licenses contain covenants
under which the licensee must agree to:
•
•
•
•
Royalties and/or milestone payments
Exclusive or non-exclusive, with various restrictions on field of use
(often) Grantback of improvements to licensor
(often) Assistance to licensor in maintaining patent monopoly
BIOS -compliant IP licenses will instead contain
covenants under which the licensee must agree to:
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• No royalties, only costs of maintaining protected commons
• Non-exclusive only
• Sharing of improvements and technology data for regulatory
purposes
• No assertion of improvement patent rights against other
licensees
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BIOS licenses will be granted to entities that
agree to the covenants:
• Universities
• Public good research institutions
• Private companies, small, medium or large, wanting to use
and improve the technology to make products
The intent of the improvement-sharing
and non-assertion requirements
is that no one licensee can hijack the
technology, and it can be used
- for humanitarian purposes
or
- to make a profit
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The
GusPlus™
project
Funded by the Rockefeller Foundation and Horticulture Australia
A new ß-glucuronidase gene
Tuan Nyugen, Peter Wenzl, Brian Weir, Heidi Mitchell, Leon Smith, Richard
Jefferson
BIOSTM licensing
Draft License: Mat Berman (UC) Mike Rabson, Marie Connett Porceddu,
Richard Jefferson; Commentable website: Steve Irwin, Nick dos Remedios;
to be jointly hosted with Science Commons
BioForgeTM distributive collaboration website
Collabnet® and CAMBIA’s BIOS Initiative