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=Alginate-chitosan-alginate=
=Module 2 Ideas=
==Encapsulation==
===Colanic acid exopolysaccharide===
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Colanic acid | Colanic Acid]]
<br>


==Specifications of encapsulation==
===Alginate Encapsulation===
These include:
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate | Alginate Encapsulation]]<br>
1) protection against low pH
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate Biosynthesis | Alginate Biosynthesis]]<br>
2) attachment in intestines
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate Properties | Alginate Properties]]
3) efficient release of the bacteria within the gastrointestine
<br>
4) use of materials that are inexpensive, stable, and of food grade
===Xanthum Gum===
5) Inducibility
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Xanthan Gum| Xanthan Gum Possibilities]]
and possibly
6) protection against oxygen, heat, and other environmental stresses during drying, formulation, and storage
 
 
==Microencapsulation with alginate==
These capsules are primarily composed of alginate, a naturally occurring polysaccharide<br>
composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. <br>
 
Calcium ions are used to cross-link G-rich regions of the alginate chains, and the resulting calcium alginate (CaAlg) <br>
hydrogel beads are coated with crosslinkers to strengthen the bead surface and control permeability. <br>
A final coating with alginate is applied in order to hide the PLL from the host(4) and make the capsules biocompatible.<br>
 
Microencapsulation has been applied to enhance the viability of probiotic bacteria during processing <br>
and also for targeted delivery to the gastrointestinal tract.<br>
 
Benefits: <br>
*mild gelation conditions
*biocompatibility
*biodegradability
*nontoxicity
*pH dependency
 
 
===Method===
 
Bifidobacterial cells were centrifuged and added to alginate solution.<br>
These were extruded to 0.1M calcium chloride through the end of a blunt needle using compressed air<br>
The cross-linking material were added.<br>
The beads were gently stirred and hardened for an hour.<br>
 
Spheres of diameter 1.5mm formed<br>
- corresponded to previous study that gel diameters of 1-3mm needed to protect bifidobacteria<br>
 
Various materials can be used for coating:<br>
polydextrose, soy fibre, skim milk, yeast extract, kappa- carageenen, chitosan and whey protein<br>
 
 
===Acid resistance===
 
Exposed to simulated gastric juice pH 1.5<br>
 
in one paper, skimmed milk  exhibited highest resistance<br>
 
[www.angelfire.com/ex/ltc/BAL.pdf / testing different materials]
 
 
Proposed reason:
 
The increase of the carboxy charges of polymeric matrices serves to <br>
- neutralise acid and enhancing the buffering effects<br>
This improved gastric stability of entrapped bacteria<br>
 
moderate protection (22–26 %) afforded by native alginate
beads seems related to the availability of D-mannuronate
carboxylate groups to intercept proton access
 
[http://www.hindawi.com/RecentlyAcceptedArticlePDF.aspx?journal=ijps&number=617184/ properties of alginate beads]
 
 
 
===Attachment===
 
The mechanism of sustained drug release is attributable to the fact that alginate is a mucoadhesive polymer; <br>
the enhanced gastrointestinal residence time is likely to be responsible for the improvement in drug bioavailability<br>
 
The ability of chitosan to modulate the intestinal tight junctions is an added virtue, which helps the encapsulated drugs <br>
in crossing the permeability barriers<br>
 
 
 
===Release===
 
[http://jac.oxfordjournals.org/cgi/content/full/53/4/635/ usage as anti-tuberculosis drug carriers]
 
nominal release (less than 7% of the encapsulated drug) in the SGF throughout the 72 h study period.
 
In the SIF, the release of rifampicin was less (16%) compared with isoniazid (20.6%) or pyrazinamide (22.1%) in the initial 6 h. <br>
Subsequently, there was a slow but sustained release of each drug, limited to less than 3% of the encapsulated drug <br>
 
Drugs encapsulated in alginate–chitosan microspheres attained Cmax at 24 h as against 1 h in the case of orally administered parent drugs. <br>
In case of free drugs, the Cmax was achieved instantaneously.<br>
<br>
<br>
The sustained release allows a reduction in dose/dosage frequency


Control:
==Sporulation==
polycationic macromolecules such as chitosan not only stabilize the alginate microspheres but also control the porosity of alginate<br>
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.<br>
to enhance the sustained release effect<br>
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Sporulation | Sporulation]]
 
Proposed reasons:


A decrease in the pH leads to shrinkage in the alginate gel and a reduced permeability of the alginate–chitosan microspheres.<br>
== Second Capsule ==
In a neutral/alkaline medium, the interpolymeric complex swells and disintegrates to release the drugs, <br>
[[IGEM:IMPERIAL/2009/Encapsulation/Phase2/Outer_Capsule]]
assisted by the sequestration of calcium ions by the phosphate present in the SIF<br>


When pH is lowered below the pKa values of d-mannuronic and l-guluronic acid (3.6 and 3.7, respectively) <br>
alginate is converted to alginic acid with release of calcium ions<br>


====Naturally Occuring Trehalase Production ====
--[[User:David Roche|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.
<br><br>
[http://www.ecocyc.org/ECOLI/new-image?type=PATHWAY&object=PWY0-1182 Link to Relavent Website]<br>
<i> 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.


===Inducibility===
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 ].


=Sporulation=
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. </i>
<br?


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.<br>
====Naturally secreting exo-polysaccharide====


Biofilm formation <br>


===<b>Sporulation and E.coli</b>===
Modify: bacteria can coat themselves


E.coli is non-sporulating. Therefore the idea to clone the genes for sporulation from Bacillus subtilis into E.coli was investigated. A paper (below) used the amino-acid sequence deduced from the nucleotide sequence of the spolIAC gene of Bacillus subtilis which has been shown to be homologous to that of the sigma subunit of the Escherichia coli RNA polymerase. Results show that this gene can be cloned in E. coli <b>only</b> under conditions in which it is not expressed.
Function: defence<br>


[http://www.springerlink.com/content/h8t0648547330465/fulltext.pdf E.coli and sporulation.]
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.


The sigma-like products of the sporulation gene spolIA C of Bacillus subtilis is toxic to Escherichia coli.<br>
Biofilm formation is triggered naturally by glucose and osmotic levels.
The main chassis we would therefore use is B.subtilis as it is the easiest to manipulate and well characterised.<br>


[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=202258&blobtype=pdf/ paper]


[http://mic.sgmjournals.org/cgi/content/full/150/8/2727/ paper 2]


It is now agreed that spore formation in bacteria is a form of differentiation in which there is an ordered, temporal sequence of events and a degree of commitment.<br>
====Alginate-chitosan-alginate coating====


Bacteria will produce a single endospore internally. The spore is sometimes surrounded by a thin covering known as the exosporium, which overlies the spore coat. The spore coat, which acts like a [sieve] that excludes large toxic molecules like lysozyme, is resistant to many toxic molecules and may also contain enzymes that are involved in germination. The cortex lies beneath the spore coat and consists of peptidoglycan. The core wall lies beneath the cortex and surrounds the protoplast or core of the endospore. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs, that protect the spore DNA from UV radiation and heat. The core also contains normal cell structures, such as ribosomes and other enzymes, but is not metabolically active.
Alginate and chitosan are two naturally occurring polymers.<br> Alginate is an anionic polysaccharide composed of mannuronic acid and guluronic acid residues, which is extracted from seaweed.<br> Chitosan is a cationic polysaccharide obtained from partial deacetylation of chitin, the main constituent of the crustacean skeleton.<br> These polymers are:<br>
*nontoxic<br>
*biodegradable<br>
*biocompatible<br>
*easily modified through physical or chemical methods.<br>
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.


===<b>The Sporulation Process & Genes Involved At Each Stage</b>===


Sporulation in Bacillus subtilis is a fertile system for studying development because of the detailed genetic understanding of this process. A summary from the paper is shown, identifying the key steps in the sporulation process.
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


1. Entry into sporulation
Alginate-Chitosan-Alginate
*Spo0A, which in its phosphorylated form stimulates expression of early sporulation genes and inhibits the expression of genes whose function is to prevent sporulation.
Cross linking alginate allows better retention of the drugs to be secreted.
*SinR is required for the development of competence and motility, but inhibitory for sporulation and the production of exoproteases.
ACA is most widely studied microcapsules for its good biocompatibility and the low cost of chitosan
*SinI binds tightly to SinR, thus preventing SinR from binding to promoter DNA.


=====Method=====


2. Final commitment
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.<br>
*SpoIIAB can bind to sigmaF and prevent sigmaF from combining with core RNA polymerase and transcribing from sigmaF directed promoters.


<b>Preparation of Beads </b>


3. Spore coat synthesis and assembly
1) Bead-Forming Solutions <br>
*GerE is the latest acting of four regulatory factors (sigmaE-SpoIIID-sigmaK-GerE) which ensure the correct temporal appearance of gene products in the mother cell.
*GerE is a 74 residue DNA-binding protein that acts both as a repressor and as an activator of expression of a number of coat proteins. Its function is to control the synthesis and assembly of the spore at the level of transcription.
*SpoIVA induced by sigma E and controls the assembly of a ring of CotE proteins.
*Coat protein genes (eg CotG) induced under the control of sigma K and the DNA-binding protein GerE.


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


===Control, Genes Involved and Potential Solution?===
2) Bead Formation by Cross-Linking and Coacervation


There are a number of transcription factors that regulate each stage of the sporulation process. Each has a number of operons that it influences.
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.<br>
The formed beads were allowed to harden for 30 min and then rinsed with distilled water.<br>
Native and functionalized chitosan coating solutions, respectively, for 1 h under gentle stirring at room temperature.<br> Beads were dried overnight at 20 °C and 40–50% RH.


=====Resistance to acidity=====


At the sporulation initiation stage, Spo0A controls 62 operons (found using [http://dbtbs.hgc.jp/ DBTBS]). The exact function of each gene is not known. However, there have been various studies done on isolating and mutating a specific gene to find the effect it has on the coat formation produced.<br>
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). <br>
Therefore, the mechanism known is more of a 'linked' pathway and the role of all individual genes is not known.


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


Therefore, instead of finding the minimum number of genes required for each stage, an idea we came up with was to keep as many genes as possible and add in an inducible promoter for production of the initial stage of sporulation. Therefore, we can control Spo0A without subjecting it to nutrient starvation processes. This can be done in a plasmid in vitro and then inserting this into the cell.<br>
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 <br>


Since the forespore will be the part producing the spore (as the mother cell lyses), it is important for our protein produced and contained within the forespore. As a result we would try to target the sigma factors that are only expressed in the forespore to produce the protein we require to transport. An example is sigma G. For this a promoter needs to be found that acts on sigma G, and the protein gene placed under the control of this promoter. The next steps in the sporulation process will be the same.
====Membrane Preperation for Alginate coating====
Inspiration from [http://www.jbc.org/cgi/reprint/282/16/11827.pdf|this] 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.
<br>


However, the process produces a number of extracellular and intracellular proteases that may degrade the protein we are trying to deliver. For this purpose, the genes involved in the production of protein degrading enzymes has been investigated (see paper below).


*So if we want to control the initial step we would need to modify the chromosome, which in ''B.subtilis'' is more feasible than in other chassis such as ''E.coli''. Tom and Chris have recently ordered a ''B.subtilis'' sporulation deficient strain, might be interesting to read how this was done.
*Id be interested to hear more of a comparison about the physical properites of the spore vs the other encapsulation methods you have looked at. Tianyi has outlined some key features that need to be met, e.g. resistance to pH as low as 2, stable at body temperature


===Useful Links===
====Storage of encapsulated cells====
[http://www.freepatentsonline.com/WO2008017483.html Link to a patent describing the surface display of enzymes on spore proteins]
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.




[http://www.ysbl.york.ac.uk/projects/1/1.2.htm Structural studies of Key Regulators of Bacterial Sporulation]
=====Sporulation=====


=Encapsulation Trigger=
*'''[[User:Chris D Hirst|Chris D Hirst]] 17:31, 20 July 2009 (EDT)''':[http://jb.asm.org/cgi/content/full/186/7/2212?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=subtilis&searchid=1&FIRSTINDEX=1100&resourcetype=HWFIG Stochastic Decisions leading to sporulation or alternative forms of action in ''Bacillus subtilis'']

Latest revision as of 08:54, 6 August 2009

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
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