Exeter/5 June 2013

From 2013.igem.org

Exeter iGEM 2013 · Paint by Coli


Dr. Nic Harmer led a discussion between the team members on synthetic polysaccharides and insulators. He worked with the 2012 iGEM Team last year; their project worked on polysaccharide formation, and by running through what they did and how their project worked, he hoped to give us an insight into iGEM and how we could use the Registry and structure of the competition to our advantage. Our notes are below.

Synthetic polysaccharide and insulators

Polysaccharides

  • Biological polymers formed from sugars, just include C, O, H usually in a specific ratio
  • More oxygen rich than most biological molecules
  • Much more difficult to work with than protein/nucleic material (difficult to purify)
  • Genetically determined, but more complex to determine sequence from DNA/RNA
  • General formula CxH2xOx in a ring structure
  • Lots of chiral centres, most 6C sugars have 4 chiral centres, so 16 variants on structure
  • Chiral centres important for 2ndry structure and tertiary structure

Synthesis

  • Common features include a precursor molecule (eg. UDP-glucose, link between phosphate and sugar contains some energy, and UDP group gives better specificity for enzymes)
  • Yeasts tend to use fatty precursor molecules, less specificity (most yeasts/higher organisms make polysaccharides in Golgi body)
  • Enzymes take sugar off of nucleotide/fatty group and attach to another sugar (occasionally non-sugar molecule)

Location

  • Provide stability for cell and cellular surfaces (cell wall, lipopolysaccharides, capsular polysaccharides, glycosylation. In higher organisms, plant cell wall, extracellular PS)
  • Gram –ve bacteria need lipopolysaccharides; removal usually leads to cell death. Also use a capsule of polysaccharides to protect from host immune system, metals and aggressive proteins. Capsules dictate a bacteria’s virulence and survival in the air/soil/water
  • Glycosylation usually increases solubility
  • Over half of all human proteins are glycosylated. Affects solubility and function of protein.
  • Interaction between cells (signalling) can use polysaccharides.

Uses

  • Vaccines produced 30 years ago were mainly based on serial reinfectionof cells with bacteria until it’s considered non-aggressive. Also boiling until inactive, but has large side effects. Many vaccines aren’t especially effective.
  • New method is to take polysaccharides from organism and inject, or something vital to the pathogen’s function (proteins/enzymes/polysaccharides). Pathogens with unusual polysaccharides are an issue; usually very dangerous pathogens, and requires a large culture to be grown to get a small amount of vaccine material.
  • Polysaccharides have unusual properties; high tensile strength, O rich so have lots of internal structure (eg. hyaluronic acid which is used in human connective tissues, so little flexibility and odd physical properties).
  • Can only collect 5/6 polysaccharides in sufficient quantities to be useful in material science
  • Long term drug release – instead of having an injection every few hours, inplant a device which slowly releases drug over a week or so. Have to use polysaccharides to enable release mechanism and gradual degradation
  • “Humanising” proteins – proteins grown in cancer cells (antibody cancer therapies), but very expensive and polysaccharides between kingdoms are very different and having the wrong polysaccharides lead to a massive immune response, rendering protein useless. If we make a large quantity of the human precursor, we can dramatically reduce the cost of the overall drug.

The problem

  • We can easily synthesise DNA and proteins (although proteins can’t be massive). CAN’T do the same with polysaccharides
  • Chemically, we have many reactive groups (generally ~5, only 2 in DNA and proteins, where they bind to make chains) which are very similar. Spend most of time stopping reactions on unwanted sites.
  • Polysaccharides are often non-linear, which is WAY more complicated
  • Synthesising a short polysaccharide takes £10,000s and many months

Synthetic Biology solution

  • Enzymes only react one enantiomer
  • We can make the reactions SCALABLE (if it works in a petri, we can scale it up to make kilograms very easily)

We need to be testing in vitro to get a silver medal, and have it planned into our schedule!!

Synthetic insulators to create a closed environment in a cell (eg. viral particle, can be assembled in E. coli and have holes to allow required substrates and DNA). Requires complex scaffolding when building to allow correct function and structure.

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli