Switch-a-roo: engineering a photoresponsive `E.colight switch`

Switch-a-roo: engineering a
photoresponsive ‘E.colight switch’
Team Macquarie University
Sydney, Australia
What are the elements
of ‘switch-a-roo’?
 Phytochrome
 Protein that binds
chromophore
 Chromophore
PDB 1ZTU
 Molecule that reacts to light
 Light
 660-800 nm range
biliverdin
Mechanics of the Light Switch
PDB 209C
INACTIVE
Biliverdin +
Bacteriophytochrome
HIGH ratio of light absorbed in
red light range (660-730nm)
ACTIVE
HIGH ratio of light absorbed
in far-red light range (730800nm)
What were our
bacteriophytochromes?
 Four domains
 Agrobacterium tumefaciens
 Able to transfer and
integrate DNA into host
genome
 Deinococcus radiodurans
 Extremely radiation
resistant
PDB 209C
Heme Oxygenase makes biliverdin
 Biliverdin produced from the catabolism of heme by
Heme Oxygenase 1
 ALA (δ-aminolevulinic acid) is a precursor in the
biosynthetic pathway of heme
heme
biliverdin
What we planned to achieve
Assemble an on/off light switch
mechanism
 Consisting of a T7 promoter, HO and
bacteriophytochrome parts.
Construct BioBricks:
 Heme oxygenase
 D. radiodurans - phytochrome
 A.tumefacians – phytochrome
 T7 promoter
Final construct
Starting materials for BioBrick construction
 Used the materials from MQ 2010 effort
Gene product
Template
RBS + HO-1
pET3a-HO1
RBS + A.tumefacians
phytochrome
pGemT-easy
RBS + D.Radiodurans
phytochrome
Amplified PCR product
BioBrick construction using RFP BBvector
 RFP BB (BBa_J04450)  Red colony
 Successful insert  White colony
 Easy visual analysis
Ladder
HO-PCR
product
HO1-BB construction
 HO amplicon
 ~800bp
0.5kb
After BioBrick
construction
 Initial screen
 Digest with XbaI
1. Uncut plasmid
2. RFP
1
2
3
4
3. Uncut plasmid without
insert
4. XbaI digest of plasmid
Our first BioBrick!
Plasmid
backbone
2.2kb
 Double digest of EcoRI and SpeI
 Shows correct orientation
 Sequencing
2kb
1kb
0.8kb
HO
fragment
BioBrick Assembly of HO + T7
T7
 T7 promoters
used:
 BBa_I712074
from 2007
Ljubljana
Team.
 BBa_I719005
from 2007
Imperial Team
HO
BiIiverdin! The HO works
 Transform into BL21(DE3) E. Coli
Contains T7 RNA polymerase
How we improved a BioBrick
 Our new HO BioBrick improves part
BBa_K497005
 Their part lacks a RBS
 Added RBS & proved functional
BBa_K646000
Successful PCR of phytochromes
 Amplicon size  2.3kbp
AT-Bph
DR-Bph
2kbp
2kbp
Next steps for our light switch
 Construction of the phytochrome BioBricks
 Removal of internal restriction sites
 Complete assembly of the light switch
Next
T7
HO1
Switch-a-roo
Bph
How is this useful?
 Switch on using red light
 Response time
 Regulation of gene expression by light
 Blood detector (assuming cell free system)
 Add team photo
Thanks for listening!