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