Bacterial Relay Race

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Bacterial Relay Race

• Anthony Tascone, Nicholas Linn
• Penn State iGEM 2005

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Thought Process
Our Idea Bacterial Relay Race ?A motile
bacteria moves along a microchannel producing a
diffusible quorum signal that, when it encounters
a second immotile bacteria, turns on a switch
controlling the latters motility, while
concomitantly switching off its own. The first
bacteria constitutively produces a diffusible
quorum molecule which the second, immotile
bacteria utilizes as an input for its switch
(initially in the off state). When the first
bacteria approaches sufficiently close to the
second, the switch will be flipped to an on
state that controls motility. Additionally, the
on state produces a second type of quorum
molecule that feeds back on the first bacteria
signaling it to stop.
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Thought Process (cont.)

• First Problem How do we turn on motility?

Solutions
The Che System ? knocking out parts of the system
creates tumbling/running mutants we want
completely non-motile bacteria
motB ? Block and Berg showed that originally
non-motile bacteria (motB-) were able to swim in
only 10-20 minutes after introduction of
functional motB gene
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Thought Process (cont.)
Second problem how to guide the cells through a
track?
Whitesides Berg made things easier
(hopefully) Build it and they will come E. coli
will spontaneously swim along the right side of
a microchannel
Berg, Whitesides, et al. E. Coli swim on the
right. Nature, 435, June 30, 2005.
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Thought Process (cont.)

• The microchannels cannot be just straight lines
• The second group of bacteria must wait for the
  first at a relay point inside of the channel
• The second cell group must be delivered to the
  microchannel
• The first cell group needs to have swarming
  capability to crawl into the channel from agar
• The relay point must be designed such that the
  first cell group will not run past the second
  before it has been turned off and the second
  group has been turned on
• Solutions
• A large (1cm x 1cm) open central chamber
• Trapping cells on a loop
• A filter or grating which blocks cells but not
  quorum sensing signals

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Thought Process (cont.)
Thrid problem how to stop the first cell group
from moving?
Solutions
The parB System ? parB contains hok/sok proteins.
Turning on expression of the hok protein by
itself would kill the cells in minutes, stopping
movement
Mutant motB ? It is possible to mutate MotB via
quickchange of a single nucleotide such that it
is non-functional but still dominant over normal
MotB. Turning on expression of mutant MotB would
stop cell motility.
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BioBrick Cloning
Cloning was carried out using the BioBrick
standard assembly method. Parts are ordered
from MITs Registry of Standard Biological Parts
and assembled using the scheme shown to the
right.
With this strategy, two parts can be assembled
using only 4 restriction enzymes Eco R1, Xba I,
Spe I, and Pst I. Since Spe I and Xba I leave
the same sticky ends, these ends may be ligated
together, forming a new site not recognized by
any restriction enzymes.
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Laboratory Procedures
Biobrick
EcoRI
XbaI
SpeI
PstI
Suffix Insertion
Prefix Insertion
PstISpeI
PstIXbaI
b. Heat Inactivation/ Phosphatase treatment
a. Gel Purfication
P
P
Ligation
Transformation
Mini-prep, restrict, sequence, Iterate
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BioBrick Assembly

• We constructed a genetic switch that responds
  to quorum sensing molecules.

The switch is divided into BioBrick parts by
dotted lines. These parts were assembled using
the methods in the prior slides.
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Cell 1 Circuit Diagram
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Cell 2 Circuit Diagram
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Future Work

• Experiment with motB- induction
• Transform motB plasmid into motB- strain plate
  cells
• Carry out chemotaxis assay with cells
• Induce with IPTG Look under microscope for
  motility

• Study conjugation and motility of F cells in
  microchannels. Rather than using quorum sensing,
  cells could tell each other to start and stop by
  transferring a DNA sequence between each other by
  conjugating.

• Fabricate master for PDMS mold
• Test additional track designs

• Mathematical modeling of genetic circuit
  behaviorthis will allow us to fine tune our
  design by suggesting changes in promoter or RBS
  strength