Biosensing with magnetotacticbacteria

1. Biosensing with magnetotacticbacteria

2. 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) »

3. 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

4. General idea Definition of the substrate will define which receptor to use!!! Any idea?????? Specific 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 Magnetosome formation 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

5. Cell receptors instead of a ligand 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 Cell that have a given allele for the drug receptor Drug Drug presenting receptor (MHC like receptor) Bacteria Signaling cascade and turning on magnetosome formation

6. How turning on magnetosome formation: two putative targets Fig. Arakaki et al, 2008 • Two options: • Inhibition of MagA or MamJ inhibitor (to be found) • Homologous recombination of MagA or MamJ

7. 1) Inhibition of inhibition MagA/MamJ inhibitor Input: binding on the receptor (ABC transporter 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

8. 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/Lectures_&_Old_Exams/Lecture_11.pdf

9. 2) Homologous recombination (Genetic circuit) 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. Input: binding on the receptor (ABC transporter * Magnetite/vesicles can be formed Inducible promoter MagA/MamJ gene Recombined genomic bacterial DNA MagA/MamJ protein

10. ABC transporter and Signaling cascade to turn on genes 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)

11. 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. !!!

12. Magnetisation of a magnet Initial magnet Hext Retentivity

13. 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

14. Read out(1) Grouping of the bacteria in a given homogeneous magnetic field Optimized boxes containing bacteria Uniformly spread bacteria Coil Bacteria form a spot in the center once they have turned on “magnetosomal” genes “By default” read out, bacteria are spread No ligand sensed Ligand sensed

15. 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

16. Read out(2) Ferromagnetic material whith a section S Measurement of the coil inductance (with N spires) sample δ

17. Read out(2) Initial inductance (without magnetite) : L0 = N² ΛΛ = μ0 S / δ If we have a Volume Vb of magnetite in samples’ volume Χequ = Vb / Vsample · Χmagnetite L = L0 · (1 + Χequ)

18. 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

19. 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?) !

20. 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

21. 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

22. Uniprot - Kegg • MagA: http://www.genome.jp/dbget-bin/www_bget?mag:amb3990 • MagA: 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