IGEM:MIT/2005/Receiver 1: ToxR

Receiver Device #1 -- ToxR

POC

wiki-Will

Function

To dimerize in the presence of the input signal and cause gene expression to turn on.

Device Depiction

Information path ...

1. Fluoroscein introduced to system (as Fluoroscein+DNAspacer+Fluoroscein), travels into periplasm.
2. Fluoroscein binds to adjacent ToxR'-scFv fusions.
   1. Causes ToxR' dimerization
3. GFP expressed @ CTX_promoter
   1. As a function of ToxR dimerization

People path ...

1. Maxine gives us Fluoroscein, or identifies something else
2. Jenny's scFv binds to Maxine's antigen
3. Then, Jenny's scFv induces ToxR' to dimerize
4. The dimerization leads to the expression of Jessica's output @ CTXpromoter [C1, maybe]
5. Ray makes the whole system look like "we meant to do that."

Device Parts

ToxR'-scFv, CTX-GFP, ToxR'-malE, Toxr'-PhoA, oligo-flour

Current Status

1. Going from DNA sequence information to physical genetic material that works inside living cells.
   1. see device parts for what we need ...
2. Getting Signal Into Periplasm
   1. CAN WE GET OLIGO's AND RUN TESTS NOW ???
3. All parts specified
   1. and maybe ready for synthesis ...

PhoA Primer.

fwd: 5'     CATGAG  GAATTC GCGGCCGC T TCTAGA ATGAAACAGAGCACCATTG 3'
             junk    EcoRI    NotI     XbaI       Primer

rev: 5'     ATGCCA  CTGCAG GCGGCCGC T ACTAGT TTATTA TTATTTCAGGCCCAGCGCC 3'
             junk    PstI    NotI      SpeI   Stop        Primer

PhoA Middle(1)

fwd: 5' caccgcggaactgcaagatgcgaccccggcg 3' 
                       *
rev: 5' CGCCGGGGTCGCATCTTGCAGTTCCGCGGTG 3'
                       *  

PhoA Middle(2)

fwd: 5' aggcttttttctgcaagtggaaggcgcgagc 3'
                       *
rev: 5' GCTCGCGCCTTCCACTTGCAGAAAAAAGCCT 3'
                       *

malE Primer.

fwd: 5' CCGTTA  GAATTC GCGGCCGC T TCTAGA ttacttggtgatacgagtct 3'
         junk    EcoRI    NotI     XbaI       Primer

rev: 5' ACTGAG  CTGCAG GCGGCCGC T ACTAGT TTATTA TGAAAATAAAAACAGGTGCA 3'
         junk    PstI    NotI      SpeI   Stop        Primer

Open Issues/Questions

• !

1. Can we get the signal in the periplasm?
2. Or, can we adapt or extend the ToxR receiver device so that it accept a signal at the outer membrane.

1. ToxR/scFv Fusion - Can we know with any more certainty that the fusion will work?