IGEM:MIT/2005/Input: Ligand

POC

Maxine

Function

• 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.

Device Depiction

Until I have time, here is a rough sketch:

Fluorescein molecule 1 =====double-stranded DNA======= Fluorescein molecule 2

Device Parts

• 2 Fluorescein molecules
• DNA primers
• original DNA strand (of varying lengths)

Current Status

Steps to take

1. Figure out if/how the ligand can pass through the outer membrane of the E.coli cell
2. Understand the content and length of the DNA that is between the fluorescein molecules
3. Learn specifics of primer design
4. Decide which company to order primers from (e.g. idtdna) and find out specifics of ordering
5. Determine if RE sites should be added into the piece of DNA (for negative testing of dimerization)

Current Work

Trying to figure out if/how the ligand can pass through the outer membrane of the E.coli cell

Troubling Data:

• 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)
• 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

Perhaps the ligand is linear enough (i.e. it has a small cross-sectional area) that even though it is a bit too heavy, it can still fit through the outer membrane?

Data collected so far regarding this hope:

• DNA specs:
• 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

Also working on looking around for other types of inputs

Possibilities:

1. scFV that binds to isoketal adducts Paper Sequence

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

• 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?

Working List of Information I've Found

(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)