Download Biosensors

Biosensing with
magnetotacticbacteria
Match with several ideas
2) « Bacteria as a diagnostics tool for still uncurable diseases: sensor for a
molecule/hormone/... specific of a disease, and would then release a substance
which could create a visible phenotype: could allow early detection of diseases
(ideally) (Gabriela) »
8) ”Create a biosensor for CO (carbon monoxyde), that would turn red or green if the
threshold of toxic CO is reached. Use of dried E. coli cells and a GFP cassette : an
inducible promoter sensitive to CO and then the GFP gene so that the E. coli cells
would turn green if there is too much CO. This could be then detect and associated
with some sort of an alarm. (Caroline)”
10) « An application in personal medicine : some treatements are efficient in patient
with a certain allele while it has no effect (or an adverse response) in the patients
with the other allele. An alternative to sequencing every patient would be to
engineer some cells so they bind to the receptor of the drug, and would fluoresce
only if not bound. Use of an inducible promoter to make the control (all cells
fluoresce at the beginning and when we add the ligand no more fluorescence if it is
bound). (Caroline) »
Which spiece?/strain?
 Magnetospirillum magneticum
 Strains : AMB-1or MGT-1
 « They are capable of growing under both microaerobic
and aerobic conditions in liquid or (DeLong et al. 1993;
Meldrum et al. 1993b), which makes them ideal
candidates for genetic manipulation ( Matsunaga et al.
1992). »
Arakaki et al, 2008
Definition of the
substrate will
define which
receptor to use!!!
Any idea??????
General idea
Specific receptor (ABC Transporter) for the ligand of interest OR
protein (type MHC protein f.ex) that carry a given ligand => *
Signaling cascade
Magnetosome
formation
Turn on magnetosome formation:
-Produce an inhibitor of the inhibitor
for a crutial protein in the
magnetosome formation pathway
-Turn on a crutial gene in the
magnetosome formation pathway
Plasmid expressing constitutively LacZ => blue cells
and expressing constitutively the receptor (ABC
transporter & other genes maybe)
* In presence of a given cell type with a given allele of a receptor if the drug bind to the receptor =>
gene activation => positive read out, if drug doesn’t bind => negative read out
Cell receptors instead of a ligand
Cell that have a
given allele for the
drug receptor
If cells coming from a patient have the
receptor for a given drug, Magnetobacteria
will respond.
=>so the patient have the good allele for this
drug
If cells coming from a patient doesn’t have
the receptor for a given drug,
bacteria doesn't respond.
=> The patient haven’t the correct allele to
be treated with this given drug
Drug
Drug presenting receptor (MHC
like receptor)
Bacteria
Signaling cascade and turning on
magnetosome formation
How turning on magnetosome
formation: two putative targets
Fig. Arakaki et al, 2008
Two options:
1)Inhibition of MagA or MamJ inhibitor (to be found)
2)Homologous recombination of MagA or MamJ
1) Inhibition of inhibition
Input: binding on the
receptor (ABC transporter
MagA/MamJ
inhibitor
MagA/MamJ
Inhibitor of
MagA/MamJ
inhibitor
*
Inducible
promoter
Magnetite/vesicles can be formed
Plasmid
To be found:
MagA or Mamj inhibitor => this will then
determine which protein will be transfected in
the bacteria
Gene that codes for a protein (that produces/which is)
an inhibitor for MagA/MamJ inhibitor
2) Homologous recombination
(based on the “single-strand invasion model”)
Steps:
- Put MagA/MamJ gene with inducible
promoter in front (a resistance to kill bacteria
that haven’t integrated DNA, and a toxin to
kill bacteria that have randomly integrated
DNA => no recomb. bact. Are killed) in a
plasmid.
- Put this plasmid in the bacteria
- Select the recombined strain
http://biology.bard.edu/ferguson/course/bio310/Lecture
s_&_Old_Exams/Lecture_11.pdf
2) Homologous recombination
(Genetic circuit)
In this recombined strain, under normal conditions MagA/MamJ is not
expressed.
Input: binding on the
Once the ligand bind, MagA/MamJ is expressed -> turn on
receptor (ABC transporter
magnetite/vesicles formation -> bacterial response.
*
Inducible
promoter
Magnetite/vesicles
can be formed
MagA/MamJ
gene
Recombined
genomic bacterial
DNA
MagA/MamJ
protein
ABC transporter
and
Oldham, M.L., Davidson, A.L., Vhen, J. (2008) Structural insights into ABC transporter mechanisms. Curr. Opin.
Struct. Biol. 18(6): 726-733 PMID:18948194.
Two options:
- the substrate induces promoter
- The signaling cascade induces promoter (this cascade may be changed a little bit)
Signaling cascade to
turn on genes
Link between the receptor (ABC
transporter) and gene turning on
Signaling cascade:
The ligand that will be used will define the ABC transporter (or another
kind of transporter) and the signaling cascade we can use/modify to
transmit signal.
!!! If the bacteria already have the receptor (ABC transporter), we just have to
make the inducible promoter respond to a specific component of this given
receptor signaling cascade. !!!
Magnetisation of a magnet
Initial magnet
Hext
Retentivity
Magnetite in Bacteria
Fe3O4 or Fe3S4 cristal
Single domain, like a magnet
But the particle are so small that in the
bacteria the magnetite are not coherent
(the bacteria can’t be considered like a
magnet). The thermal agitation move the
direction of the magnetisation.
10-150 nm
The sum of magnetite can be
considered like soft magnetic
material (small hysteresis)
Characteristic : χr
Read out(1)
Grouping of the bacteria in a given homogeneous magnetic field
Coil
Uniformly spread
bacteria
“By default” read out, bacteria are spread
No ligand sensed
Optimized
boxes
containing
bacteria
Bacteria form a spot in the center once they have
turned on “magnetosomal” genes
Ligand sensed
Read out(1’)
Bacteria will move to where the
magnetic flux is the most important.
A coil or a magnet
The goal is to have a gradient of the magnetic
field in the sample
Read out(2)
Ferromagnetic material
whith a section S
sample
δ
Measurement of the
coil inductance
(with N spires)
Read out(2)
Initial inductance (without magnetite) :
L0 = N² Λ
Λ = μ0 S / δ
If we have a Volume Vb of magnetite in samples’ volume
Χequ = Vb / Vsample · Χmagnetite
L = L0 · (1 + Χequ)
Required components for the
read out
Read out 1 :
- Self inductance or a magnet
- But the system should be large enough
- The number of bacteria define the sensitivity of the system
Read out 2 :
- Self inductance, magnetic circuit and a small volume for the sample
- Electrical circuit to mesure the inductance
- To have a good sensitivity, we need to work in small scale
- The system is dependent of the number of bacteria
Rapid and low cost
 Fluorescence -----> detection material=too expensive, BUT
!
here LacZ -----> blue color => Nothing to measure
=> Cheap (the only thing that will cost is maintaining
bacteria)
=> Electronic material ---> cheap
 Formation of vesicles --> 15min first immature vesicles
(Staniland et al, 2007) => rapid
(further goal: optimize vesicles formation to observe the
shape in 5 min?)
Further applications
 Different strains with different ABC transporters can be
engineered => large scale screening
(?) Maybe set of XXX-well plate with XXX different
molecules that can be sensed.
=> Large scale diagnosis with rapid and easy read out.
----> In case of “allele tracking” : can screen for many
alleles (=> receptor-drug binding) in on XXX-well plate
 Can be used in medical context as well as in screening
for detox context
Limitations
 ABC transporters (but with a library of mutated
transporters ---> can be unlimited)
 Ligands maybe not possible for an infinite ligand set.
 Magnetobacterial genome not extremely well known
Uniprot - Kegg
 MagA: http://www.genome.jp/dbgetbin/www_bget?mag:amb3990
 MagA:
http://www.uniprot.org/uniprot/Q2W031#section_attribut
e
 MagA: http://www.genome.jp/dbgetbin/www_bget?uniprot+Q2W031
 MamJ : http://www.genome.jp/dbgetbin/www_bget?uniprot+Q3BKB2_9PROT