Biosensing with magnetotacticbacteri

a

Match with several ideas2) « 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 magneticumStrains : 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

General ideaSpecific receptor (ABC Transporter) for the ligand of interest OR protein (type MHC protein f.ex) that carry a given ligand => *

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

Signaling cascade

Plasmid expressing constitutively LacZ => blue cellsand 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

Magnetosome formation

Definition of the substrate will define which receptor to use!!!Any idea??????

Cell receptors instead of a ligand

Drug presenting receptor (MHC like receptor)

Drug

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

Signaling cascade and turning on magnetosome formation

Bacteria

How turning on magnetosome formation: two putative

targets

Two options: 1)Inhibition of MagA or MamJ inhibitor (to be found)2)Homologous recombination of MagA or MamJ

Fig. Arakaki et al, 2008

1) Inhibition of inhibitionMagA/MamJ inhibitor MagA/MamJInput: binding on the

receptor (ABC transporter

Plasmid

Magnetite/vesicles can be formed

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

Inhibitor of MagA/MamJ inhibitor*

Inducible promoter

2) Homologous recombination

(based on the “single-strand invasion model”)

http://biology.bard.edu/ferguson/course/bio310/Lectures_&_Old_Exams/Lecture_11.pdf

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

2) Homologous recombination

(Genetic circuit)

MagA/MamJ gene

Inducible promoter

Input: binding on the receptor (ABC transporter

MagA/MamJ protein

Magnetite/vesicles can be formed

Recombined genomic bacterial

DNA

In this recombined strain, under normal conditions MagA/MamJ is not expressed. Once the ligand bind, MagA/MamJ is expressed -> turn on magnetite/vesicles formation -> bacterial response.

*

ABC transporter

Signaling cascade to turn on genes

and

Two options: - the substrate induces promoter- The signaling cascade induces promoter (this cascade may be changed a little bit)

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.

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

Hext

Initial magnet

Retentivity

Magnetite in Bacteria

Single domain, like a magnet

10-150 nm

Fe3O4 or Fe3S4 cristal

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.

The sum of magnetite can be considered like soft magnetic material (small hysteresis)Characteristic : χr

Read out(1)

“By default” read out, bacteria are spread

Bacteria form a spot in the center once they have turned on “magnetosomal” genes

No ligand sensed Ligand sensed

Grouping of the bacteria in a given homogeneous magnetic field

Optimized boxes

containing bacteria

Uniformly spread bacteriaCoil

Read out(1’)

A coil or a magnetThe goal is to have a gradient of the magnetic field in the sample

Bacteria will move to where the magnetic flux is the most important.

Read out(2)

Measurement of the coil inductance(with N spires)

sample

Ferromagnetic materialwhith a section S

δ

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 applicationsDifferent 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

LimitationsABC 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 - KeggMagA: http://www.genome.jp/dbget-bin/www_bget?

mag:amb3990MagA:

http://www.uniprot.org/uniprot/Q2W031#section_attribute

MagA: http://www.genome.jp/dbget-bin/www_bget?uniprot+Q2W031

MamJ : http://www.genome.jp/dbget-bin/www_bget?uniprot+Q3BKB2_9PROT