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From 2010.igem.org

Beads

Why beads? make the case for encapsulation, subpages for specifics:

Team:BCCS-Bristol/Wetlab/Beads/Gellan

Team:BCCS-Bristol/Wetlab/Beads/Safety_Benefits

Team:BCCS-Bristol/Wetlab/Beads/Procedure

Team:BCCS-Bristol/Wetlab/Beads/Improvements

Team:BCCS-Bristol/Wetlab/Beads/Encapsulation_Applications

There are several reasons why the E. coli cells are encapsulated in a gel bead. The primary concerns were visibility of the fluorescent signal and being able to keep the cells separated from the environment. There are secondary benefits too, it is possible to include nutrients in the gels for the bacteria to consume and the beads can help to absorb nitrates from the soil.

Initial experiments looking at the visibility of E. coli constitutively expressing GFP on soil, e.g. fig. 2, showed that it would be difficult to observe fluorescence from E. coli simply sprayed onto soil in solution. The lab-grade E. coli are designed to be out-competed by naturally occurring bacteria and they are unable to synthesise certain amino acids. This is designed as a safety feature, it ensures that they will not survive for long outside of a lab environment. This means that when sprayed directly onto unsterilised soil the E. coli will die relatively quickly, so only those that survive will be able to sense nitrate and produce a signal. The bead provides a protective environment for the E. coli and packages a large amount of them together to produce a large signal. The bead can also be infused with a nutrient broth to provide the cells with an adequate supply of energy and amino acids to ensure survival and continued production of GFP and RFP.

Initial experiments using beads that had just the PyeaR+GFP BioBrick showed that the in- crease in fluorescence is easily detectable, before and after images can be seen in fig. 4. The beads were placed on wet unsterilised soil that had been saturated with a 20nM potassium nitrate solu- tion. There is some fluorescence in the ’before’ picture, this is due to ’leakage’ expression of GFP, i.e. the residual expression in the absence of nitrate due to the repressor not repressing every singleP yeaR site.

It is expected that the beads will limit the spread of E. coli into the field. Since the E. coli are dependant upon amino acids in their nutrient broth they should not last too long outside of the bead environment, and when they have exhausted their supply internally they should die off too. This factor of the bead design has not been rigorously experimentally tested though. It is unclear exactly how densely packed the E. coli are in the bead, and how much movement they have, preliminary data from high magnification confocal microscopy that those at the edge of the bead can move out of it quite freely.