Toxics MultiBiosensor

http://2009.igem.org/files/poster/UAB-Barcelona.pdf

The aim of our study was to design a multiple biosensor for toxic compounds present in water, taking advantage that microorganisms
are cheap, self-regenerable and it is possible to get a quick response. To achieve the challenge of having a bacteria able to detect
many compounds, we started with separate biosensors.
The first design was an E. coli that could detect trihalomethanes and, specifically, chloroform.
The second approach was to detect contamination by excess of nutrients, quantifying the presence of nitrite and phosphate.
Toxics MultiBiosensor
Students: Marina Badia, Albert Canet, Belén Garcia, Rubén Fernández,
Fátima Franco, Antonio Freire, Arturo Morales, Alberto Osuna
Advisors: Juan Antonio Baeza, Pau Ferrer, Joan Albiol
Universitat Autònoma de Barcelona. Bellaterra, Catalonia, Spain
PROJECT 1: Trihalomethanes PROJECT 2: Nitrite and Phosphate
Why trihalomethanes?
Trihalomethanes (THM), and chloroform in particular, are toxic
recalcitrant compounds. An important source of THM
is the chlorination of drinking water or swimming pool water. Fig. 1. Chloroform molecule
CHLOROFORM: Biobricks design
Sayavedra et al1 isolated 2 genes (mbla and clpB) of Nitrosomonas europaea that are
selectively induced by chloroform. In order to obtain an E. coli strain behaving as
Nitrosomonas, our idea was to co-express the N. europaea amo (ammonia
monooxygenase) encoding gene with either mbla or clpB:
In this way, depending on the amount of chloroform in the medium, we should be able to
detect more or less intensity of green fluorescence in E. coli cells.
1 Sayavedra-Soto et al. Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in
response to co-oxidation of chloroform. Appl Microbiol Biotechnol (2009)
Fig. 2. Chloroform detection
biobricks for E. coli
Results Although basal
fluorescence was
observed, no
chloroform-induced
fluorescence was
detected.
Materials and Methods
As N. europaea has a very slow growth
rate and amo isolation by PCR failed, we
therefore decided to transform E. coli with
the plasmids containing the gfp gene
under the mbla- and clpB promoters
provided by Prof. Sayavedra (that is,
without amo).
The standard GFP expression experiment
performed is described in Fig. 3. CF
concentration, induction time, growth
phase and OD were modified in an
attempt to find the best conditions.
Fig. 3. Standard incubation protocol
Kinetic modeling
The modeled system is divided into the three phases where the chloroform appears: air
phase, bulk liquid and inside the cell.
All reactions have been assumed as mass action.
Model Statements:
– GFP response is high around pH 6.
– GFP response increases with promoter concentration
Behavior as a biosensor:
The model is sensitive enough to detect initial low
concentration of chloroform (around 1 ppb), and for
values up to 20 ppb leads to a saturated response. Fig. 6. Model of the chloroform biosensor
Conclusions
– GFP basal expression in the cells with
mbla/clpB + GFP plasmids
– Addition of CF did not change this
GFP basal expression
– Both promoters are recognized by an
E. coli transcription factor
– Although E. coli has its own clpB, its
regulatory machinery can not
recognize Nitrosomonas’ one
Future work
– Introduce the amo gene in the
expression cassette
– Find transcriptional regulators
allowing induction of mbla and clpB
promoters by phosgene in E. coli
Fig. 7. Potential CF response pathway in E. coli
Fig. 5. Confocal microscopy image
of E. coli with GFP
Fig. 4. Graphics of the fluorescence emitted
Problems
PHOSPHATE: Biobricks design
phoA promoter from E. coli that is activated in absence of phosphate
pphoa
Kinetic modeling
A mathematical model of a
theoretical phosphate biosensor was
developed to study the production of
GFP and RFP, regulated by
inorganic phosphate. The model
includes:
– External phosphate transport and
across the cell membrane
– Detection of the phosphate
concentration at the cell surface
– Signal transduction cascade
– Gene regulatory network (including
activation and repression
of phoA promoter)
20 ordinary differential equations
and 1 algebraic expression were
integrated using an algorithm
suitable for stiff equations in Matlab
NITRITE: Biobricks design
nir promoter from N. europaea that is still functional and constitutive in E. coli
NSR: nitrite sensitive repressor from N. europaea, in absence of nitrite binds
to the promoter and stops transcription
pnirk
nsr
Fig. 8. Biosensor in absence of nitrite Fig. 9. Biosensor in presence of nitrite
Fig. 10. Biosensor in absence of phosphate Fig. 11. Biosensor in presence of phosphate
Materials and methods
– PCR isolation and cloning of required
elements from N. europaea and E. coli
DH5α.
– Selection of required existing biobricks
– “Cut and paste” to make the new
constructs = biobricks we want
New regulatory system to be
used for researchers!!!
NsrR (nitrite sensitive repressor)
+ pnirK
pnir is constitutive in E. coli
It is repressed if NSR is produced!
With the addition of NO2
– to the
medium, NSR is released and the
synthesis begins.
pnirk
nsr
Accomplishments
– Cloning of all genetic elements
– Submitting BioBrick Bba_K248001
(nsrR)
We gratefully acknowledge the support of the Universitat Autònoma de
Barcelona and the Department of Chemical Engineering at UAB

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: