27
Light-Based Cell-Cell Signaling
VERSATILITY
PENETRABILITY
ORTHOGONALITY
CONTROL
Introduction Sender Cell Receiver Cell Human Practices Discussion
Renilla Luciferase
CMV PromoterBBa_I712004
Renila
CMV PromoterBBa_I712004
470nm Photons 470nm Photons
Coelenterazine
HEK 293T Cell
HEK 293T Cell
CatCh-eYFP
Humanized Aeq
BBa_K548000
eYFP
Ca2+
Introduction Sender Cell Receiver Cell Human Practices Discussion
Our System
Humanized Aeq
BBa_K548000
Renilla Luciferase
CMV PromoterBBa_I712004
Renila
CMV PromoterBBa_I712004
470nm Photons 470nm Photons
Coelenterazine
HEK 293T Cell
HEK 293T Cell
CatCh-eYFP
eYFP
Ca2+
The Sender Cell
Introduction Sender Cell Receiver Cell Human Practices Discussion
Gaussia and Renilla Luciferase
Coelenterazine + O2
http://profmother.files.wordpress.com/2010/06/seapansy3_jpg.jpg
http://www.cmarz.org/CMarZ_RHBrown_April06/images_press/index.html
Coelenteramide + CO2
+ 470 nm photons
Gaussia orRenilla
Introduction Sender Cell Receiver Cell Human Practices Discussion
Ivis Lumina II
2.79 E5
5.53 E65.53 E6
2.79 E5
pRL-CMV-Renilla
pRRL-CMV-Gaussia
Introduction Sender Cell Receiver Cell Human Practices Discussion
0 100 200 300 400 500 600 7007.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8
8.1
Time (s)
Radi
ance
x 1
0^8
Renilla + CZ Luminescence Time
Course
Introduction Sender Cell Receiver Cell Human Practices Discussion
Humanized Aeq
BBa_K548000
Renilla Luciferase
CMV PromoterBBa_I712004
Renila
CMV PromoterBBa_I712004
470nm Photons 470nm Photons
Coelenterazine
HEK 293T Cell
HEK 293T Cell
CatCh-eYFP
eYFP
Ca2+
The Receiver Cell
CatChAequorin
Introduction Sender Cell Receiver Cell Human Practices Discussion
pRRL–CMV–Aeq
+ + =
Aeq
pENTR-d/TOPO-Aeq
pENTR5-CMV
pRRL
pRRL-CMV-Aeq
Introduction Sender Cell Receiver Cell Human Practices Discussion
Characterization of Aequorin
Aeq HEK Aeq HEK with Ca
Aeq HEK with CZ
Aeq HEK with Ca + CZ
1E+01
1E+02
1E+03
1E+04
1E+05
1E+06
1E+07
1E+08
Rela
tive
Lig
ht U
nits
Introduction Sender Cell Receiver Cell Human Practices Discussion
Fura 2-AM Ca2+ Imaging Ratio-metric Calcium
Dye
Emission – 510 nm
Calcium Free Excitation – 380nm
Calcium Bound Excitation – 340 nm
Measure change in emission at 340/380
Introduction Sender Cell Receiver Cell Human Practices Discussion
380 nm 340 nm
340/380
Fura 2-AM Ca2+ ImagingIonomycin – Positive Control
Introduction Sender Cell Receiver Cell Human Practices Discussion
Fura 2-AM Ca2+ Imaging
Introduction Sender Cell Receiver Cell Human Practices Discussion
pRRL-CMV-CatCh-eYFP
Fura 2-AM Ca2+ ImagingpRRL-CMV-CatCh-eYFP co-cultured with pRL-CMV-Renilla
Introduction Sender Cell Receiver Cell Human Practices Discussion
Human Practices
http://www.dailymail.co.uk/sciencetech/article-1279988/Artificial-life-created-Craig-Venter--wipe-humanity.html
http://www.preciouslife.com/saimages/346.png
Introduction Sender Cell Receiver Cell Human Practices Discussion
Goals
Introduction Sender Cell Receiver Cell Human Practices Discussion
1. How pervasive is the media’s influence on public perception of Synthetic Biology?
2. What can Synthetic Biologists do to ensure that the public has a balanced view of the field?
Conducted a survey of over 200 people
Asked a wide variety of questions
http://www.aseonline.biz/Portals/0/survey-man.jpg
Introduction Sender Cell Receiver Cell Human Practices Discussion
How Big is Media Influence?
Biology-Related Field
Non-Biology Related Field
Non-Classroom Biology
Experience
Introduction Sender Cell Receiver Cell Human Practices Discussion
How Big is Media Influence?
Heard of Synthetic Bio
Heard of Genetic Eng.
Not Heard of Either
Introduction Sender Cell Receiver Cell Human Practices Discussion
How Big is Media Influence?
38% News Media (non-scientific)
28% Science-focused media
19% Scientific Literature
Other Sources 14%
Introduction Sender Cell Receiver Cell Human Practices Discussion
How Big is Media Influence?
How Big is Media Influence?
44% News Media (non-scientific)
25% Science-focused media
12% Scientific Literature
Other Sources 19%
Introduction Sender Cell Receiver Cell Human Practices Discussion
What Can We Do?
http://bioethics.gov/
http://bioethics.gov/cms/valerie-bonham
Valerie Bonham
Introduction Sender Cell Receiver Cell Human Practices Discussion
“The media shapes perception” “We need to recognize, respect, and
understand the fear surrounding Synthetic Biology”
Synthetic Biologists need to join the conversation!
We are not heard in the most widely disseminated forms of media.
Optimization of interactions between sender & receiver
cells
Renilla Luciferase
CMV PromoterBBa_I712004
Renila
CMV PromoterBBa_I712004
470nm Photons 470nm PhotonsCoelenterazine
HEK 293T Cell
HEK 293T Cell
CatCh-eYFP
Humanized Aeq
BBa_K548000
eYFP
Ca2+
Introduction Sender Cell Receiver Cell Human Practices Discussion
Discussion
Our sender cell can produce a detectable
signalOur receiver cell can respond to bright light
Looking Ahead…Precoelenterazine (PCZ)
Interfacing with existing biobricks
Introduction Sender Cell Receiver Cell Human Practices Discussion
Thanks Everyone!
Dr. Casim SarkarDr. Jordan MillerDr. Dan Cohen
Dr. Anthony
Choo
Daphne Ng Najaf Shah
Questions?
