finding needles in haystacks - university of...
TRANSCRIPT
14 July 2017 P1789-P-153 v0.3
Finding needles in haystacks
A high-throughput platform for genetic construct screening
Richard Hammond
Synbio control engineering conference 17 July 2017
14 July 2017 P1789-P-153 v0.3 2
Who we are
Technology consultants and product developers
14 July 2017 P1789-P-153 v0.3 3
What we do
From start-ups to multinationals – engineering biology
Business strategy
Wetware development
R+D processes and tools
Manufacture processes
14 July 2017 P1789-P-153 v0.3 4
Characterisation platform need
Massively-ParallelMultiplexed Microfluidic Assays
Microfluidic droplet-based
14 July 2017 P1789-P-153 v0.3 5
Characterisation platform need
Wetware development – microorganisms as biosensors
Hexavalent chromium detection in water
Cr concentration
Ou
tpu
t si
gnal
Dynamic range
Limit of detection
Sensitivity
Saturation limit
B0034 T0002chrB
p_chrBR0040 [E.coli]ElvJ L3S2P21B0034RiboJ YFP
14 July 2017 P1789-P-153 v0.3 6
Separate into drops < 1
tranformant / drop
DROPLETS
Culture and let
transformed cells grow
Screen and separate
transformed cells
Competent cells and DNA post transformation
(Millions)
Waste
Array out cells from store
and expose to analyte
(~10,000)
Incubate cells to get
response
ARRAYED
Measure response and compare to requirement
Put transformed
cells into store(~1000)
Choose cells with best
performance
[]
Res
po
nse
Desired response
Characterisation platform answer
Next-generation characterisation platform concept
14 July 2017 P1789-P-153 v0.3 7
Characterisation platform – selection and characterisation
Droplet-based selection: high speed, sequential process
Poisson distribution – cells / drop = 1/htransformation
Dropletize, grow out, select
Array-based characterisation: low speed, parallel process
Highest possible density (experiments / unit area)
Simple method of exposing cells to analyte
Fourier-transform
spectrometer
50k drops/second
Droplet arraying
system
14 July 2017 P1789-P-153 v0.3 8
Miniaturised characterisation
300µm
60µm
12mm
16m
m
Miniaturisation of characterisation assay
Use of microarray spotting techniques to deposit ~10nl of bacterial
culture onto ultra-flat agar substrate containing analyte
Initial efforts focused on 8x12 (x96) sample grid to replicate
microplate assays, ran with continuous sampling in plate reader.
Compare ‘micro-colony’ device performance on agar to liquid
culture characterisation assay in 96-well plate
1.5mm
o/n growth
14 July 2017 P1789-P-153 v0.3 9
Miniaturised characterisation
Macro-scale fluorescence detector and ultra-flat agar
Custom-built optics to give
required field of view
Custom-built moulds for agar
pouring
– Defined location in X and Y
– Defined height
– Mould glued to glass slide
below, avoid bubbles through
air bleed
– Scrape second slide as knife-
edge across the top of the
mould
14 July 2017 P1789-P-153 v0.3 10
Miniaturised characterisation
Repeatable pinning out… and sectoring of colonies
Sample
GFP single-channel constructs#1 #2 #3
ColonyColony Colony
Device #1
Device #2
Device #3
14 July 2017 P1789-P-153 v0.3 11
Miniaturised characterisation
Analyte-in-agar; IPTG induction of pTAC-YFP system
Reference Standard (Rows 1 & 8);
pCC-1789-116 (Rows 2,4,5,7);
pCC-1789-232 (Rows 3 & 6);
-ve
Co
ntr
ol
1u
M
10u
M
50u
M
pCC-1789-232 (Row 1)
pCC-1789-116 (Row 2);
Reference Standard (Row 3);
14 July 2017 P1789-P-153 v0.3 12
Miniaturised characterisation
Analyte-in-agar; IPTG induction of pTAC-YFP system
-ve Control 1uM 10uM 50uM 100uM
Reference Standard (Column 1); pCC-1789-116 (Column 2); pCC-1789-232 (Column 3)
14 July 2017 P1789-P-153 v0.3 13
Miniaturised characterisation – data analysis
Algorithm for extracting dose response (1mM data shown)
Calculate local maxima of DI
Raw image
ID Find edgesCreate watershed map, filter to find
circles
Align centres to known 12 x 8 array,
fill in gaps
Colony intensity
Normalise for background
1 2 3 4 5
1
2 3
2 4 4
5
14 July 2017 P1789-P-153 v0.3 14
Miniaturised characterisation – data analysis
Dose response data
Reference Standard
(row 0, 7)
pCC-1789-116
(Row 1, 3, 5, 6)
pCC-1789-232
(Row 2, 4)
The technique provides repeatable measurement of dose response from
multiple colonies
14 July 2017 P1789-P-153 v0.3 15
Miniaturised characterisation – next steps
We intend to push this technique further
Comparison to liquid assay in plate reader
– Demonstrate comparable response and limit of detection
Ratiometric measurement
– Two fluors, one controlled by a standard constitutive promoter, one by the
genetic circuit of interest
– Normalise out common-mode noise
– Characterise deltas through genetic circuit variation only
High-throughput massively parallel characterisation then supports methods
such as automated parameter inference to characterise and measure genetic
circuits accurately
14 July 2017 P1789-P-153 v0.3 16
A high-throughput platform for genetic construct screening
Many thanks to the team
Rodrigo
Zapiain
James
Brown
Sam
Pooley
Tom
Collings
Sara
Dodd
Doug
O’Rourke
Alan
Sanders
This work was self-funded by CCL. Patent application number 1709413.7
Au
tom
atio
n
Op
tics
Sig
na
l pro
ce
ss
ing
We
t lab
Bio
log
ica
l sa
fety
Pro
jec
t ma
nag
em
en
t
Sys
tem
arc
hite
ct
14 July 2017 P1789-P-153 v0.3
UK
Cambridge Consultants is part of the Altran group, a global
leader in Innovation. www.Altran.com
www.CambridgeConsultants.com
USA SINGAPORE JAPAN
Registered No. 1036296 England