IGEM:MIT/2005/Input: Ligand

POC

http://www.ci.berkeley.ca.us/environmentalhealth/images/bacteria.gif
Maxine

Function

• To design an input for receiver unit 1 (ToxR) and receiver unit 2 (FecA)
  • Receiver 1:
    • To design a input (ligand) with 2 fluorescein molecules attached by a piece of DNA, which will be used in the intermediate step of testing if binding of an antigen to our system can cause dimerization and subsequent transcription of the desired output gene.
  • Receiver 2:
    • Input here is simply just a fluorecein molecule to test if binding of an antigen to our system can cause a conformation change and subsequent transcription of the desired output gene.

Device Depiction

http://www.steve.gb.com/images/molecules/phthalates/fluorescein.png Fluorescein Structure

Device Parts

Lengths: 10 bp 12 bp 15 bp 20 bp

• A pair of ssDNA strands complementary to each other, each bound to a fluorescein molecule at the 5' end, of various lengths.

Current Status

Completed Work

1. Determined the length and content of DNA between fluorescein molecules
   1. 10 bp ~ 33.2 A: because DNA twists every 33.2 A, at this point, the fluorescein molecules, on different strands of DNA, will be pointing directly away from each other, 180 degrees apart
   2. 12 bp ~ 39.2 A: fluorescein molecules will be pointing 90 degrees apart
   3. 15 bp ~ 50.2 A: fluorescein molecules will point in the same direction
   4. 20 bp ~ 66.4 A: fluorescein molecules will point 180 degrees away from each other
2. Obtained sequence of puc19 and used it for DNA construct
3. Ordered from Invitrogen, with TK's help--should arrive Friday, Jul 15

Steps to take

1. Figure out if/how the ligand can pass through the outer membrane of the E.coli cell
   1. Make cell wall more permeable with electroporation, heatshock cells, or find cells with mutation in the outermembrane??
   2. Find other types of inputs that are small enough to fit into the cell
2. Determine if RE sites should be added into the piece of DNA (for negative testing of dimerization)

Current Work

• Figure out if/how the ligand can pass through the outer membrane of the E.coli cell

• Troubling Data

cell membrane premeability ~ 600 Daltons ~ 6 amino acids Source

DNA base pair average molecular weight ~ 650 Dalton Source and Source

Fluorescein: 330 daltons

Also, see "DNA specs" under "Maxine's Notes" Section

• Possible Solutions

• Is ligand linear enough (i.e. it has a small cross-sectional area) to fit through the outer membrane despite its large mass?

Fluorescein dimers for Epo receptor: best linker ~ 45 A

DNA diameter: ~26 A

"Antisense agents are 10 or more bp, and this length is typically too large for efficient passive cellular uptake by diffusion across lipid bilayers" Source

• Use other types of inputs

NPN as possible replacement for fluorescein? Paper [[../NPN/]]

Nitrocefin as poss replacemeht for fluorescein? Paper [[../Nitrocefin/]]

scFV that binds to isoketal adducts Paper Sequence

Digoxin: 780 Da -- too heavy

• Increase cell wall permeability:

• Mutation in outer membrane: "Lipoprotein Mutation Accelerates Substrate Permeability-Limited Touluene Dioxygenase-Catalyzed Reaction," Biotechnology Progress (article not available online--see Maxine for copy of printed version)
• Attachment of cell-permeabilizing peptide to our input Paper [[../PNA Paper Review/]]
• Electroporation
• EDTA treatment
• Heatshock

• DNA transporter: bacterial conjugation--too complicated??

Input Reception

Questions

1. Is our fluorescein dimer entering into the cell?
2. Distance in space between flurs. -- might depend on 3D conformation/wobblyness
3. Separate out single from double stranded

Experiments

1. Lets throw the oligos in, wash the media, look for flur. and we can hope that means its diffused
   • control: normal fluor. -- don't touch the oligos just yet
   • READOUT: can we see it under a microscope? get antibody -- into cytoplasm? selectivly trash outer membrane? will's oligo thingy?

Open Issues

• Is fluorescein too big to get into the periplasm?
• Important to note that the optimal distance of 12/13 mer was for the EpoR protein.. ToxR might have a different optimal distance.

Need Help With

Issue: can we ignore the fact that our construct is too many Daltons by assuming that according to the geometry, our construct is linear enough to pass through the outer membrane?

• Find people with experience w/cell membrane permeability of e. coli
• Find different way to link fluorescein molecules

Maxine's Notes

(this section is purely for myself so that I have a spot to put my own work) [[../Maxine's Notes/]]