SWITCHING ON THE POWER OF E. COLI
Lydia Alldred, Aimee Bias, Martin Campbell, Jake Casson, Robbie Evans, Amy Ferguson, Beth Grieg, Gemma McLelland , Jacob Roberts, Gintarė Sendžikaitė
MOTILITY
ABSTRACT
We created a basic model of
bacterial swimming (based on a
Random Walk) for use in
investigating the effects of gas
vesicles. This led us to knock out
swimming, as detailed below.
We aimed to create a heritable system that, in the presence of a specific
stimulus, would switch between expression of one set of genes (bacterial
motility) and another (gas vesicles) based on the site-specific
recombinase, φC31 integrase. We envisioned many possible applications,
but decided to focus on water purification. We prepared bacterial
knockouts for two flagellar genes and successfully rescued the mutants
using our motility biobricks. This switch was shown to work through the
use of GFP and RFP on opposite sides of the switch, using the sugar
arabinose as the trigger in our proof-of-concept system.
100 step directed
“Random Walk”
Swarm assays to test motility of KO and WT strains. On 0.3%
agar, E. coli can swim across the surface. If motile bacteria are
spotted onto the centre, they migrate outwards; non-motile
do not. Both fliC and motA KOs were totally defective in
swimming, whereas the WT strains could swim to the edges.
Gene rescue of fliC restored swimming to WT level, ΔmotA
required motA/motB to swim.
WT
FLOTATION
THE SWITCH
Φc31 Integrase binds to attP and attB sites in DNA. It creates
double strand breaks, inverts, and then ligates the DNA, creating
attR and attL sites which can no longer be cut. Our idea was to
make Integrase expression inducible, to allow cells to switch from
expressing swimming genes, to genes for producing gas vesicles. As
this is a change to the DNA, it is heritable and irreversible.
During our two visits to
Glasgow Science Centre, we
were able to interact with
the public and gauge their
opinion on our project.
They enjoyed our poster,
and the kids loved our
bubble-based-bonanza.
We calculated a range of
floating speeds for vesicle-filled
bacteria. Adding a buoyancy
component to our random walk
had little effect on natural
movement – swimming needed
to be knocked out.
BEST BIOBRICKS
BBa_K1463000
Our switch
BBa_K1463560
Our reverse RBS
BBa_K1463602
Our fliC/promoter
construct
POLICY AND PRACTICE
We focused on water purification for our switch.
Freshwater accounts for 2.5% of the world’s water and with
increasing water shortages it is more vital to make the other
97.5% available. We asked the public their opinion on uses
for bacteria-purified water, and they strongly favoured use
in agriculture as opposed to drinking.
Gas vesicles are biconical protein
compartments (right above) made
from gvps (gas vesicle proteins) that fill
with surrounding gas, allowing
cyanobacteria to float. When we
transformed gvps into E. coli, they
arrested cell division (right below).
This picture shows the above construct with GFP and
reverse RFP (under the control of our reverse RBS)
When the cells were exposed to arabinose, this induced
Integrase, and the cells stopped expressing RFP, and
instead expressed GFP. Left is exposure to glucose, right
is exposure to arabinose. Overlay of red and green
fluorescent images using 532 nm laser and LPG filter
and 473 laser and BPB filter respectively
Thanks to Dr Sean Colloms, Dr Julien Reboud, Professor Marshall Stark, Ms Zhao Jia, Ms Emma Smith, Ms Abioye Jumai
FUTURE WORK
• Test integrase under the control of different inducible
promoters
• Create functioning gas vesicles
• Test function of motility and vesicle genes within
inversion switch
• Attempt aggregation using University of Aberdeen’s
AG43 to increase flotation velocity.
Dillon,R. Fauci,L. Gaver,D. A Microscale Model of Bacterial Swimming, Chemotaxis and Substrate Transport.Journal of Theoretical
Biology.1995. Vol 177(4). p 325-340. Walsby, AE. Gas Vesicles. Microbiological Reviews. 1994. Volume 58. p 138.