TM
A new selectable marker geneA new selectable marker gene
TheThe GUSPlus GUSPlusTMTM project project
A new GUS gene, Available under BIOSTM licensing,
Pioneering use of the BioForgeTM concept
TM
Why do we want extra-cellular GUS?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
TM
GUSPlusGUSPlusTMTM is a more sensitive reporter than is a more sensitive reporter than E.coliE.coli GUS GUS
E.coli GUS vs GUSPlus activity in trans-activator facilitated enhancer trap lines - inferred from X-glucA staining intensities
0
5
10
15
20
25
30
35
40
45
50
no expression weak medium strong
Expression level/Staining intensity
% transformants
E.coli GUS GUSPlus
Rice anthers and style showing GUSPlusTM
expression
Properties of Properties of GUSPlusGUSPlusTMTM
√ 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
TM
Synthetic Synthetic GUSPlusGUSPlusTMTM gene constructiongene 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
TM
GUSPlusGUSPlusTMTM has lower Khas lower Kmm, similar V, similar Vmax max toto E.coli E.coli GUSGUS
1/[pNPG] (mM -1)1/[pNPG] (mM -1)
-20-20 -10-10 00 1010 2020
1/v
(n
mol-1
·g·m
in)
1/v
(n
mol-1
·g·m
in)
0.010.01
0.020.02
0.030.03
0.040.04
BGUSBGUSEGUSEGUS
-1/Km-1/Km
1/Vmax1/Vmax
BGUS EGUS Km 40 M 120 M
Vmax 80 80 nmol.g -1min-1
TM
GUSPlusGUSPlusTMTM has enhanced activity, and has enhanced activity, and improved thermal stabilityimproved thermal stability
Time (min)Time (min)
00 1010 2020 3030
Temperature (°C)Temperature (°C)
00 2020 4040 6060 8080
Rela
tive a
ctiv
ity (
%)
Rela
tive a
ctiv
ity (
%)
00
2020
4040
6060
8080
100100
GUSPlusGUSPlusE.coli GUSE.coli GUS
60°C60°C
TM
GUSPlusGUSPlusTMTM and and E.coliE.coli GUS have similar, GUS have similar, broad pH optima broad pH optima
pHpH
55 66 77 88 99
Act
ivit
y (
nm
ol·g
-1·m
in-1)
Act
ivit
y (
nm
ol·g
-1·m
in-1)
00
2020
4040
6060
8080
100100
GUSPlus GUSPlus E. coli GUSE. coli GUS
TM
00
2020
4040
6060
8080
100100
120120
5 m
M G
lcA
5 m
M G
lcA
10 %
DMSO
10 %
DMSO
10 %
DMF
10 %
DMF
1 %
For
malde
hyde
1 %
For
malde
hyde
0.5
% S
DS
0.5
% S
DS
1 %
Trit
on X
-100
1 %
Trit
on X
-100
1 %
Sar
cosy
l
1 %
Sar
cosy
l
1 M N
aCl
1 M N
aCl
Rela
tive a
ctiv
ity (
%)
Rela
tive a
ctiv
ity (
%) GUSPlusGUSPlus
E. coli GUSE. coli GUS
Assayed with 0.25mM pNPGlcA
GUSPlusGUSPlusTMTM has has improved chemical improved chemical resistancesresistances
TM
GUSPlusGUSPlusTMTM with signal peptide is secretedwith 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
TM
New possibilities with a secreted GUSNew 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 glucuronide-conjugated effector molecules
• Use as screenable marker gene
• Use as selectable marker gene
TM
How will this technology be available for use How will this technology be available for use by the agricultural R&D community? 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
TM
GusPlusGusPlusTM is available for use is available for use under the conditions of a BIOSunder the conditions of a BIOSTM license license
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
BIOSTM-compliant IP licenses will instead contain covenants under which the licensee must agree to:• 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
TM
The intent of the improvement-sharing The intent of the improvement-sharing and non-assertion requirements and non-assertion requirements is that no one licensee can hijack the is that no one licensee can hijack the technology, and it can be used technology, and it can be used - for humanitarian purposes - for humanitarian purposes or or - to make a profit - to make a profit
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 The GusPlusGusPlus™ project project
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
Funded by the Rockefeller Foundation and Horticulture Australia
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