IGEM:IMPERIAL/2009/Encapsulation/Phase2

Module 2 Ideas

Encapsulation

Colanic acid exopolysaccharide

Colanic Acid

Alginate Encapsulation

Alginate Encapsulation
Alginate Biosynthesis
Alginate Properties

Xanthum Gum

Xanthan Gum Possibilities

Sporulation

An endospore is a dormant, tough, and non-reproductive structure produced by Gram-positive bacteria. They form through the production of an encapsulating spore coat within the spore-forming cell. Examples include Bacillus and Clostridium.
Sporulation

Second Capsule

IGEM:IMPERIAL/2009/Encapsulation/Phase2/Outer_Capsule

Naturally Occuring Trehalase Production

--David Roche 06:14, 5 August 2009 (EDT) One thing to think about is the fact that some bacteria (incl E. coli K12) produce periplasmic trehalase to break down trehalose into glucose under high or low osmotic conditions (perhaps the growth media?). Could be a problem, but looking about a bit, it seems that it is also part of the self-preservation mechanism.

Link to Relavent Website
While E. coli only synthesizes trehalose under conditions of high osmolarity, it can degrade the sugar under conditions of both low and high osmolarity. In fact, E. coli can grow with trehalose as the sole carbon source. Different pathways are employed under different osmolarity conditions.

Under high osmotic conditions the bacterium synthesizes large amounts of trehalose, which is used as an osmoprotectant [ Giaever88 ]. Trehalose molecules that leak from the cytoplasm into the periplasm can be recycled by the action of the TreA, a periplasmic trehalase. TreA breaks trehalose into two glucose molecules, which are then recycled by transport back into the cytoplasm through the glucose PTS [ Styrvold91 ]. Another function of TreA is the utilization of external trehalose under conditions of high osmolarity [ Boos87 ].

A second trehalase, which is cytoplasmic (encoded by the treF gene), is active during the transition period between high and low osmolarity. As the cells are shifting their metabolism to adjust to low osmolarity, TreF removes the internal pool of trehalose. The relatively low enzymatic activity of TreF is low enough not to compromise the biosynthesis of trehalose during high osmolarity, yet is sufficient to degrade the accumulated trehalose after the return to normal conditions, when no more biosynthesis occurs. <br?

Naturally secreting exo-polysaccharide

Biofilm formation

Modify: bacteria can coat themselves

Function: defence

Another important trait that fortifies biofilm resistance is the sticky matrix which may contain DNA and other polymers but in general, is predominantly composed of exopolysaccharides.

Approximately 6 genes including algC are required for alginate synthesis. They all plays a significant role in Pseudomonas aeruginosa biofilms.

Biofilm formation is triggered naturally by glucose and osmotic levels.

paper

paper 2

Alginate-chitosan-alginate coating

Alginate and chitosan are two naturally occurring polymers.
Alginate is an anionic polysaccharide composed of mannuronic acid and guluronic acid residues, which is extracted from seaweed.
Chitosan is a cationic polysaccharide obtained from partial deacetylation of chitin, the main constituent of the crustacean skeleton.
These polymers are:

• nontoxic
  
• biodegradable
  
• biocompatible
  
• easily modified through physical or chemical methods.
  

They are widely used in encapsulation applications due to their ability to form gels in the presence of certain divalent cations such as calcium, barium, and strontium by ionotropic gelation.

Alginate encapsulation: Alginate is negatively charged, so it does not bind well with the negative charge of the cell membrane. (as opposed to Calcium ions) -Might be difficult to induce negative charges like calcium encapsulation

Alginate-Chitosan-Alginate Cross linking alginate allows better retention of the drugs to be secreted. ACA is most widely studied microcapsules for its good biocompatibility and the low cost of chitosan

Method

Beads were formed by ionotropic gelation via calcium cross-linking and by alginate−chitosan complex coacervation, and the effect of the polymer modification on their microstructure was also examined.

Preparation of Beads

1) Bead-Forming Solutions

Two core bead solutions and two coating solutions were prepared.
Core bead solutions contained 1.5% of WPI and 3% of native or functionalized alginate

2) Bead Formation by Cross-Linking and Coacervation

Each core bead solution was dropped through an 18-gauge needle (38.1 mm length, 1.8 mm diameter) from a 60 mL plastic syringe into a beaker containing a calcium chloride solution (10%, w/v) under gentle stirring, at room temperature.
The formed beads were allowed to harden for 30 min and then rinsed with distilled water.
Native and functionalized chitosan coating solutions, respectively, for 1 h under gentle stirring at room temperature.
Beads were dried overnight at 20 °C and 40–50% RH.

Resistance to acidity

entrapment in alginate microspheres with diameters of 40–80 μm resulted in insignificant protection of bifidobacteria during exposure to simulated gastric juice at pH 2.0 (Truelstrup Hansen et al., 2002) while larger alginate (1–3 mm) microspheres protected entrapped cells (Lee & Heo, 2000).

few studies report on the use of these polymers for delivery of encapsulated bacteria.

Simulated gastric juice (SGJ) was prepared by dissolving pepsin in saline (0.2% NaCl, w/v) to a final concentration of 0.3 g l−1 and adjusting the pH to 2.0 with concentrated HCl

Membrane Preperation for Alginate coating

Inspiration from [1] article

Protein sequence with a high calcium binding affinity has been characterised. GGXGXDXUX (X, any amino acid; U, large hydrophobic amino acid). In this way we could express this sequence on the surface membrane of our cell (on the fimbria/ membrane proteins etc), and then this would give a calcium coating to the cell. alginate could then be added to the solution, and this should form a capsule around the cell.

Storage of encapsulated cells

Our cells must be resistant to dessication. E.coli contain the otsBA genes (trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB)) which code for the enzymes required for trehalose synthesis. Trehalose provides powerful dessication resistance and therefore it might be worth up-regulating these genes prior to encapsulation. Note, since glucose-6-phosphate is the feedstock for trehalose production, it would reduce flux through the alginate synthesis pathway.

Sporulation

• Chris D Hirst 17:31, 20 July 2009 (EDT):Stochastic Decisions leading to sporulation or alternative forms of action in Bacillus subtilis