User:Healy

Harnessing the adhesive power of C. crescentus in a standard biological part

We would like to propose further investigation of the adhesive mechanism of C. crescentus, which was recently shown to be the strongest biological adhesive known. Access to an adhesive would be invaluable in many applications of synthetic biology if the functionality could be isolated genetically and its properties quantified ex vivo (outside of its natural host). One important area of research is to test the properties of oligomers of p-GlcNAc, which is found at the binding site in these bacteria and has been shown to be crucial to their binding strength. We also would like to research viable methods of attaching this system to a subject of interest, such as E. coli.

• Tsang et al. Adhesion of single bacterial cells in the micronewton range. PNAS 103 (15): 5764. (2006)

The adhesive properties of C. crescentus are quantified in this paper. Through several experiments it was discovered that the holdfast system in this bacterium has an adhesive strength of 68 N/mm^2, the strongest biological adhesive ever characterized. In fact, in most of their experiments the bacterial stalk broke before the holdfast-substrate bond. It was demonstrated that the presence of GlcNAc polymer was required for the extreme bonding strength, but the mechanism by which GlcNAc functions as an adhesive is unknown.

• Janakiraman and Brun. Cell Cycle Control of a Holdfast Attachment Gene in Caulobacter crescentus. J. Bacteriology 181 (4): 1118. (1999)

The holdfast is a conglomerate of polysaccharides that is attached to the bacterium by a gene family, named HfaA-D. This gene family is activated during the cell cycle, with the maximum level acheived during the predivisional stage. Additionally, it was discovered that the holdfast is attached to the base of the stem shortly after daughter cells begin stem synthesis.

• Nierman, W. et al. Complete genome sequence of Caulobacter crescentus. PNAS 98 (7): 4136-4141. (2001)

The genome of C. crescentus has been completely sequenced; there are 3767 genes composed of approximately 4 Mbp of DNA. This is useful reference for obtaining the sequences of DNA to be used in the future standard biological part.

• Li, G., Smith, C., Brun, Y., Tang, J. The Elastic Properties of the Caulobacter crescentus Adhesive Holdfast Are Dependent on Oligomers of N-Acetylglucosamine. J. Bacteriology 187(1): 257-65. (2005)

The holdfast of Caulobacter crescentus is examined closely to explore its various properties, and these properties are used to show that the holdfast's properties are largely dependent on the presence of N-Acetylglucosamine polymers. For instance, the holdfast stalk's strength is independent of its length - it acts as a rigid rod; also, the holdfast exhibits gel-like behavoir (similar to N-Acetylglucosamine). When enzymes that can cleave N-Acetylglucosamine were introduced, the strength of the holdfast dropped significantly, supporting the hypothesis that its oligomers are important in maintaining the integrity of the holdfast.

Outline

1. Summary: We want to make the adhesive holdfast from C. crescentus a standard biological part to put on the Registry.
   
2. Background:
   • Info about the part in its native setting (make sure to explain that it's not just one single protein) (note that it consists of the polymer clump, and the machinery that fixes the polymer clump in the right place).
   • The "why" slide - the system is the most powerful biological adhesive known. Advertise its strength and compare its stats to other known products if possible. Briefly mention applications (eg, show small pics representing the areas in the market that would be affected).
3. The Problem: How do we separate the holdfast from its organism on the DNA level? Will involve many genes! Is the product self-assembling, or do we have to find a way to incorporate the diff. proteins appropriately? Is there a way to make it work effectively without a cell?
4. Methods (we may want to propose one or all of these studies):
   • What properties of the polymer make it have such high adhesive strength? Can we replicate it synthetically? Are all the components necessary? - if we vary length, % composition, etc., can we make different "grades" of strength for different applications? Not everyone will want epoxy-like strength.
   • Location genes known, but polymer synthesis genes not known. Possibly look for similar homologues
   • Location genes rely on the species, since they act during asymmetric mitosis. The genes must be adapted to no longer locate on the stem, but on the surface of the cell, for example. Mitotic regulation might need to be removed.
6. Results:
   • Direct applications - biological "glue." Could be used in settings when non-biological adhesives are inappropriate / yield allergic reactions.
   • Indirect: synthetic biology gains the ability to use and manipulate biofilms
7. • Resources needed: Device to measure force being applied, a method of knocking out a gene here and there in order to do the individual-component strength analysis (e.g. siRNA), genome sequences for other organisms in order to identify the polymerization enzymes (the N-glucosamine polymerization protein might for instance be found in insects that use it to make chitin).
   • Impact: Biomedical devices, esp. implanted ones; maybe general-use as well.