Team:BCCS-Bristol/Wetlab/Beads/Gellan

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==Gellan==
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<center> • [[:Team:BCCS-Bristol/Wetlab/Beads|Overview]] • [[:Team:BCCS-Bristol/Wetlab/Beads/Gellan |Bead Materials]] • [[:Team:BCCS-Bristol/Wetlab/making_beads|Making Beads]]
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• [[:Team:BCCS-Bristol/Wetlab/difference_solution|Beads in Solution]] • [[:Team:BCCS-Bristol/Wetlab/difference_soil|Beads in Soil]] • [[:Team:BCCS-Bristol/Wetlab/Beads/ImageGallery|Image Gallery]] • </center>
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Why gellan? why/how did we choose this material?
 
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=Gellan=
The beads are made out of a gellan gel. Gellan is an extracellular anionic heteropolysaccharide
The beads are made out of a gellan gel. Gellan is an extracellular anionic heteropolysaccharide
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produced by Sphingomonas elodea [16]. It is water soluble and gels ionically. The polysaccharides
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produced by ''Sphingomonas elodea'' [1]. It is water soluble and gels ionically. The polysaccharides
strongly cross link in the presence of divalent cations forming a robust gel. It is biodegradable
strongly cross link in the presence of divalent cations forming a robust gel. It is biodegradable
and safe for human and animal consumption. It is used in the food industry, often as a texturing
and safe for human and animal consumption. It is used in the food industry, often as a texturing
agent in confectionery. It has an almost identical refractive index to water, meaning that it will
agent in confectionery. It has an almost identical refractive index to water, meaning that it will
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transmit light from the fluorescent proteins effectively. It is slightly autofluorescent, though less
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transmit light from the fluorescent proteins effectively. It is slightly autofluorescent, though less
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autofluorescent than untransformed E. coli. The diffusion constant for small molecules (e.g. nitrate)
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autofluorescent than untransformed E. coli. The diffusion constant for small molecules (e.g. nitrate)
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is effectively the same as for water, the diffusion constant for larger molecules (e.g. sugars) is slightly
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is effectively the same as for water, the diffusion constant for larger molecules (e.g. sugars) is slightly
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lower, though within one order of magnitude for equivalent temperatures [17]. This means that it
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lower, though within one order of magnitude for equivalent temperatures [2]. This means that it
should help absorb nitrate from the soil and equalize its concentration in the bead.
should help absorb nitrate from the soil and equalize its concentration in the bead.
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The procedure for making the gel beads is adapted from standard procedures in cell encapsu-
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The procedure for making the gel beads is adapted from standard procedures in cell encapsulation [3]. The gellan is sourced as a powder and is mixed with deionised water and heated to
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lation [18]. The gellan is sourced as a powder and is mixed with deionised water and heated to
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90 ◦ C to form a viscous fluid. It is then cooled and mixed with a highly concentrated slurry of cells
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90 ◦ C to form a viscous fluid. It is then cooled and mixed with a highly concentrated slurry of cells
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when the temperature reaches 56 ◦ C. This mixture is then pipetted into a container of 0.5 molar
when the temperature reaches 56 ◦ C. This mixture is then pipetted into a container of 0.5 molar
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room temperature calcium chloride solution in drops of a few hundred microlitres. The process of
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room temperature calcium chloride solution in drops of a few hundred micro-litres. The process of
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gelation is rapid, the outside of the drop solidifies as soon as it hits the calcium chloride solution,
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gelation is rapid, the outside of the drop solidifies as soon as it hits the calcium chloride solution,
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creating a droplet shape with distinctive ’shockwaves’ as can be seen in fig. 4(b). The beads are
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creating a droplet shape with distinctive 'shockwaves' as can be seen in fig. 1.  
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relatively uniform in size, see fig. 5 for an example of their scale. The beads are stored in nutrient
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broth before use to ensure that the E. coli survive. In fig. 6 one can see examples of different beads
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[[image:Bead_diff.png|left|200px|thumb|Fig 1, Glowing beads imaged with a microscope]]
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The beads are relatively uniform in size, see fig. 2 for an example of their scale. The beads are stored in nutrient
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broth before use to ensure that the ''E. coli'' survive. In [[media:beads.png | this figure]] one can see examples of different beads
that have been stored, the two tubes on the right for almost 3 weeks, and the two tubes on the left
that have been stored, the two tubes on the right for almost 3 weeks, and the two tubes on the left
for less than 1 week. They appear to stay intact in solution for a period of about ten days. On soil
for less than 1 week. They appear to stay intact in solution for a period of about ten days. On soil
they become damaged if they dry out too much, but otherwise retain their shape for a period of
they become damaged if they dry out too much, but otherwise retain their shape for a period of
about a week.
about a week.
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<br \>
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<br \>
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<br \>
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[[Image:RFPonSoil.png|center|400px|frame|Fig 2, Beads on a standard petri dish]]
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[1] et al. S. Patil. Study of formulation variables on properties of drug-gellan beads by factorial
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design. ''Drug Development and Industrial Pharmacy'', 32:315-326, 2006.
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[2] et al. S. Bayarri. Diffusion of sucrose and aspartame in kappa-carrageenan and gellan gum
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gels. ''Food Hydrocolloids'', 15:67-73, 2001.
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[3] T. M. S. Chang. Procedures for microencapsulation of enzymes, cell and genetically engineered
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microorganisms. ''Molecular Biotechnology'', 17:249-260, 2001.

Latest revision as of 19:44, 27 October 2010

• Overview • Bead Materials • Making Beads • Beads in Solution • Beads in Soil • Image Gallery •


Gellan

The beads are made out of a gellan gel. Gellan is an extracellular anionic heteropolysaccharide produced by Sphingomonas elodea [1]. It is water soluble and gels ionically. The polysaccharides strongly cross link in the presence of divalent cations forming a robust gel. It is biodegradable and safe for human and animal consumption. It is used in the food industry, often as a texturing agent in confectionery. It has an almost identical refractive index to water, meaning that it will transmit light from the fluorescent proteins effectively. It is slightly autofluorescent, though less autofluorescent than untransformed E. coli. The diffusion constant for small molecules (e.g. nitrate) is effectively the same as for water, the diffusion constant for larger molecules (e.g. sugars) is slightly lower, though within one order of magnitude for equivalent temperatures [2]. This means that it should help absorb nitrate from the soil and equalize its concentration in the bead.


The procedure for making the gel beads is adapted from standard procedures in cell encapsulation [3]. The gellan is sourced as a powder and is mixed with deionised water and heated to 90 ◦ C to form a viscous fluid. It is then cooled and mixed with a highly concentrated slurry of cells when the temperature reaches 56 ◦ C. This mixture is then pipetted into a container of 0.5 molar room temperature calcium chloride solution in drops of a few hundred micro-litres. The process of gelation is rapid, the outside of the drop solidifies as soon as it hits the calcium chloride solution, creating a droplet shape with distinctive 'shockwaves' as can be seen in fig. 1.

Fig 1, Glowing beads imaged with a microscope

The beads are relatively uniform in size, see fig. 2 for an example of their scale. The beads are stored in nutrient broth before use to ensure that the E. coli survive. In this figure one can see examples of different beads that have been stored, the two tubes on the right for almost 3 weeks, and the two tubes on the left for less than 1 week. They appear to stay intact in solution for a period of about ten days. On soil they become damaged if they dry out too much, but otherwise retain their shape for a period of about a week.


Fig 2, Beads on a standard petri dish


[1] et al. S. Patil. Study of formulation variables on properties of drug-gellan beads by factorial design. Drug Development and Industrial Pharmacy, 32:315-326, 2006.

[2] et al. S. Bayarri. Diffusion of sucrose and aspartame in kappa-carrageenan and gellan gum gels. Food Hydrocolloids, 15:67-73, 2001.

[3] T. M. S. Chang. Procedures for microencapsulation of enzymes, cell and genetically engineered microorganisms. Molecular Biotechnology, 17:249-260, 2001.

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