Download Poster - iGEM 2009

Bacteria-to-Yeast Communication: Using Light as a Trans-activating Factor to
Bridge a Physically Split Canonical lac operon
A. Goyal, X. Guan, A. Raman, N. Kamath, M. Dymerska, I. Bochkov, O. Medvedik, J. Jocz, D. Thompson
Harvard University
Two-Hybrid System Plasmid Construction
ABSTRACT
Optical communication is central to interactions between many multicellular
organisms. However, it is virtually unknown between unicellular organisms,
much less between unicellular organisms of different kingdoms of life. Our
team has constructed a system that allows for interspecies, bacteria-to-yeast
optical communication. In this system, bacteria would be able to communicate
to yeast the presence of IPTG, which results in transcription of lacZ in the
yeast cells. To permit bacteria to send an optical signal, we expressed in E. coli a
red firefly luciferase under IPTG induction. To allow yeast to receive the
signal, we used a two-hybrid-system based on the interaction between the redlight-sensitive Arabidopsis thaliana phytochrome PhyB and its interacting factor
PIF3. Interaction between PhyB and PIF3 could therefore be induced by
emitted red light from the bacteria, resulting in transcription of the lacZ gene.
This is an excellent demonstration of the principles and potential of synthetic
biology: this system would enable us to optically bridge a physically separated
canonical lac operon using light as a trans-acting factor, communicated
between the species of cells using photons. In other words, we would be able
to separate de-repression and gene expression into two separate cells, bridging
this physical separation with light based signals between the cells. The bacteria
signal to yeast that the operon has been de-repressed using bioluminescence
from the luciferase enzyme. In response to this optical signal, the yeast
completes the operon’s function and expresses beta-galactosidase.
Variables involved in Bacteria-to-Yeast Experiments
Potential Experimental
Result
PCB Biosynthesis and Extraction
Phy B requires PCB in order to function. Since yeast cells do not normally express
Phytochrome B, they do not have the enzymes required to create PCB. Thus PCB had to
be added to cultures for PhyB to function. PCB is light sensitive, so its extraction required
a darkroom with chemical fume hood and green safe light that would not induce the
breakdown of the PCB.
Left: When yeast cells were
Center: Crude PCB extract
Right: PCB Biosynthesis Plasmid
grown with PCB we found that
before column purification.
w/ PcyA and Ho1
the PCB did not have any
negative effect on yeast cell
growth.
Variable Responsible
+ Control
- Control
Experiment
Poor Batch Quality of Crude
PCB Extract?
+ Control
- Control
Experiment
Low expression levels of red
luciferase in bacteria (low light
emission levels)
+ Control
- Control
Experiment
Elevated B-gal expression level of
yeast cultures (problems with
induced background expression).
One of Three Repeats Gave Positive Results
Experimental:
Bacterial Light + PCB
Negative Control: Bacterial Light
+ DMSO
BACKGROUND
In bacteria the lac operon controls expression of betagalactosidase, which breaks down lactose. Laser-based Characterization Experiments
In presence of red fluorescent light and PCB,
Beta-galactosidase is produced in our PhyB/PIF
3 system.
A filter lift assay using nitrocellulose filters to screen for Y190 colonies with 2 hybridsystem. Successful colonies re-streaked onto leucine and tryptophan deficient plates were
re-screened after overnight exposure to red light in presence of PCB
Luciferase expression under IPTG
Induction in E. coli
Transcriptional changes via PhyB-PIF3
Two-Hybrid System in Yeast (Y190) Biodot Assay (Another screen for B-Galactosidase Activity)
Immobilized cells from Y190 liquid culture were lysed with liquid
nitrogen and incubated at thirty degrees Celsius overnight in 150uL
X gal buffer
PCB Concentration Assay (Another screen for B-Galactosidase Activity)
Y190 cells with PhyB-DBD and PIF3-AD were grown to a
concentration of 10^6 cells/ml, and were plated in 100uL of
solid media with varying concentrations of PCB (10, 25, 50uM).
All tested concentrations of PCB induced lacZ expression.
. Continuation of PCB Concentration Assay
The bacterial red luciferase must emit at a wavelength that can be absorbed by the PhyB in order for the signal to be relayed to the yeast. Based on
published emissions spectra for red luciferase and absorbance spectra for PhyB, it appeared that their spectra overlapped enough that light from the
luciferase would be able to induce the conformational change in PhyB necessary to setting off the signaling cascade resulting in gene expression. Right
from Gambetta et al., Left from Branchini et al.
Laser-pettor
To expedite the laser based characterization experiments, a “laser-pettor” was designed,
built and tested. The laser-pettor allows a researcher to simultaneously irradiate up to eight
samples contained within a 96-well microtiter plate.
The circuit designed for this project is a modification of that presented on (http://
www.kpsec.freeuk.com/projects/timer.htm). Briefly, when the switch is closed the circuit
activates the ~650nm, <3mW laser diodes, which remain on for a pre-set duration. After a
time delay, they automatically switch off, a green LED turns on and a beeper sounds. The
duration of time which the laser diodes remain on is adjusted by tuning a variable resistor
and/or switching between one of three capacitors.
Laser-pettor
Laser Diode Array
Circuit Diagram
Used same concentration of cells as assay above Cells were pulsed with 650 nm
wavelength. We found that cells given a ten second pulse showed the best induction,
so we then used a ten second pulse on the cells and compared induction after
incubating pulsed cells for 30 and 60 min.
Q-Tip swabs used to determine
optimal pulse length. 10 second pulse
results in the highest level of
induction.
CONCLUSIONS
• We have succeeded in rebuilding (from scratch) the yeasttwo hybrid system published by Quail et al.
• We have determined that crude PCB extract can be used
without HPLC purification.
Dependence on Cell Culture Age
The schematic below summarizes the system we would like to create: the bacteria express red luciferase under IPTG induction (derepression of the lac
promoter), and this red light is absorbed by the PhyB/PIF3 two-hybrid system in yeast. This in turn results in expression of beta-galactosidase completing
the lac operon. The two groups of cells are separated by a polycarbonate barrier, so light is the only means of communication between the two groups of
cells.
Y190 contains beta-gal and His reporters under the control
of the Gal promoter. We found that older Y190 cells have
higher background in the negative control. We
hypothesized that this was a result of mutations upregulating histidine production because this would also
result in an increase in beta-gal production. We
supplemented our media with additional histidine to try to
fix this.
Older cells not exposed to
light show beta-gal activity
Dependence on Cell Concentration
High levels of positive background were observed with high
concentrations of cells. Dilution of a culture reduces positive
background. Cells were exposed to light according to the diagram
on left.
As we can see on the right, the top row contains the highest
density of cells, showing a more positive background
expression of beta-gal.
FUTURE DIRECTIONS
• Repeating the bacteria-to-yeast signaling experiments
• Testing the PCB biosynthesis constructs.
• Putting a different reporter under control of the system,
such as green luciferase or HO Endonculease.
REFERENCES
Branchini, Bruce R. Southworth, Tara L. Khattak, Neelum F. Michelini, Elisa. Roda, Aldo. “Red- and green-emitting firefly luciferase mutants for
bioluminescent reporter applications.” Analytical Biochemistry 345 (2005): 140-148.
Gambetta, Gregory a. Lagarias, J. Clark. “Genetic engineering of phytochrome biosynthesis in bacteria.” PNAS 98.19 (2001): 10566-10571.
Shimizu-Sato, Sae. Huq, Enamul. Tepperman, James M. Quail, Peter H. “A light-switchable gene promoter system.” Nature Publishing Group 20 (2002).