IGEM:MIT/2005/Input: Ligand

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POC

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

Device Parts

• A pair of ssDNA strands complementary to each other, each bound to a fluorescein molecule at the 5' end, of the following 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 (http://www.rzuser.uni-heidelberg.de/~u53/abstracts/macdonald.html)

DNA base pair average molecular weight ~ 650 Dalton (http://www.growtall.com/technical-data3.htm and http://www.eppendorf.com)

Fluorescein: 330 daltons

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

• Possible Solutions

• Is ligand is linear enough (i.e. it has a small cross-sectional area) that it can still 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" http://www.jbc.org/cgi/content/full/277/9/7144

• Use other types of inputs

NPN as possible replacement for fluorescein? See NPN

Nitrocefin as poss replacemeht for fluorescein? See 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: http://www.jbc.org/cgi/content/full/277/9/7144
• Electroporation
• EDTA treatment
• Heatshock

Experiments

Input reception Experiments

• Move information in link to respective place.

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)

Key Facts

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

• the permeability of the cell membrane is 600 Daltons, which is about 6 amino acids

http://www.rzuser.uni-heidelberg.de/~u53/abstracts/macdonald.html

• Fluorescein dimers were designed with linker lengths: 8 mer (27.2 angstroms) to 14 mer (47.6 angstroms)

In mammalian paper under "must read" on the main page

• Best linker: 12/13 mer (though all induced growth)

In mammalian paper under "must read" on the main page

• The average molecular weight of DNA base pair is 650 Dalton

http://www.growtall.com/technical-data3.htm and http://www.eppendorf.com

• Fluorescein: 330 daltons
• DNA specs:

Other Background Info

• Picture belongs to University of Georgia.
  • Outer membrane: This lipid bilayer is found in Gram negative bacteria and is the source of lipopolysaccharide (LPS). LPS is toxic and turns on the immune system; LPS is found in Gram negative, but not in Gram positive, bacteria.
  • Cell wall is composed of peptidoglycan (polysaccharides + protein), the cell wall maintains the overall shape of a bacterial cell. The three primary shapes in bacteria are coccus (spherical), bacillus (rod-shaped) and spirillum (spiral). Mycoplasma are bacteria that have no cell wall and therefore have no definite shape.
  • Periplasm: This cellular compartment is found only in those bacteria that have both an outer membrane and plasma membrane (e.g. Gram negative bacteria). In the space are enzymes and other proteins that help digest and move nutrients into the cell.
  • Inner membrane: also known as plasma membrane. This is a lipid bilayer much like the cytoplasmic (plasma) membrane of other cells. There are numerous proteins moving within or upon this layer that are primarily responsible for transport of ions, nutrients and waste across the membrane.
• Problems
  • The outer membrane is the major permeability barrier in Gram negative bacteria. Gram negative bacteria store degradative enzymes in the periplasmic space.
• Solutions
  • Work with E.Coli strain that is cell-wall deficient
  • Or work with E.Coli strain that has the most permeable cell wall <-- LIMITATION of antigen size. Ways to make cell wall permeable are: treatment with chemicals (example: EDTA)
  • Linkers
• Statistics:
  • Cell wall: in Bacillus licheniformis and Bacillus subtilis, a diffusional radius of not more than 2.5 nm for molecules is determined (Estimates of the porosity of Bacillus licheniformis and Bacillus subtilis cell walls)
  • Cell outer membrane: in E.Coli and Salmonella Typhimurium, molecules of 600 Da and less diffuse through.
• Peptidoglycan hydrolytic activities associated with bacteriophage virions. paper with some in depth characterization of ways around/through the periplasm. maybe we can learn something from how phages shoot through the periplasm to do their bizness. (unread)