IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Notes

Biofilm Detection: Notes

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Project Schedule

Week 3 - 4

Specific Reading:

• Diffusion of vesicles
• Concentration of AHL in biofilms
• Alternatives amplifiers to HRP

Modelling of system

Background Reading:

• General information on biofilms
• Further reading on application of system
• Suitable gels for diffusion
• Catalog search for AHL

What to do by end of Week 4 Wednesday:

• Write proper specifications
• Protocols of experiments on specific parts

Week 4 - 5

• Building basic constructs
• Testing basic constructs in E.coli
• Plan experiments with HRP and cell free system

Week 6 - 7

• Build constructs with HRP and luciferase
• Test in cell free system

On Diffusion Through The Biofilm

Effective diffusion depend on solute type and density of biofilm. Mean relative effective diffusive permeability (De/$aq) of different solutes:

• inorganic anions or cations - 0.56
• nonpolar solutes (with molecular weights of 44 or less) - 0.43
• organic solutes (molecular weight greater than 44) - 0.29

Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms. Large solutes are effectively excluded from microbial cells, small solutes partition into and diffuse within cells, and ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients)due to sorption to the biofilm matrix. (21)

When substances diffuse into biofilms, they are not distributed evenly, due to the clusters of cells within the biofilm. This provides protective pockets for biofilms, from which regrowth can occur. (22)

Notes

The metabolism of cells is different at different depths within the biofilm. At the deepest level of a mature biofilm, anaerobic respiration may predominate.

Clusters of nitrite oxidizers crowd around distinct clusters of ammonia oxidizers (20, 29) (see references above). Thus, is the metabolic waste product of the ammonia oxidizers, nitrite, made available to the bacteria that can use it as a substrate for oxidation. The activities of these commingled species lead to the consumption of ammonia and oxygen near the biofilm surface and the simultaneous production and consumption of nitrite slightly below the biofilm surface.

Initial ideas

• be able to detect that it is in the presence of a biofilm on
  • contact lenses
  • catheters (which type?)
  • surgical instruments
  • kitchen utensils
  • pipes
  • food samples
  • IUDs
• be able to adsorb to, and penetrate, the biofilm
• survive long enough in the biofilm in order to disperse it (probably using DspB)
• be able to disperse 99% of the biofilm material

• Parameters to quantify
  • maximum time for biofilm dispersal
  • minimum amount of biofilm to be dispersed
  • environmental conditions for system to work in (temperature, pH, pressure)
  • any others..?

On Biofilm Formation

Biofilm Formation

Biofilm Formation

There are three stages of biofilm formation:

1. The initial attachments of microorganisms to a surface.
2. After a certain density is reached a 'slime capsule' is built up. This capsule is composed of exopolysacchorides that are secreted from the cell.
3. The final stage is the growth of the biofilm to form the distinct architecture that is associated with biofilms. Biofilms are composed of layers, much like our own skin, these layers has very specific architecture.

Toggle Switch Summary

Toggle Switch is interfaced with a transgenic Quorum signaling pathway that detects AHL and transcribes a gene which inhibits the growth of biofilm when cell population reaches a threshold density. We seek to use this for keeping the amount of biofilm at a maximum level. In addition to stopping growth the toggle switch will also turn the biofilm green when the threshold is reached.

Adhesion Problems

Factors Affecting Bacterial Adhesion:

• Biological:
  • Osmolarity & Growth Phase: OmpZ/OmpA
  • Membrane Stress : Cpx pathway & Rcs Pathway

• Physical:
  • Hydrophobicity of surface
  • Roughness of surface
  • [PVC is easily bound to - Harvard1998]

Destroying biofilms

The process of biofilm investigation carried us through a host of different-natured biofilms, including those produced by MRSA and Pseudomonas Aeruginosa. The scope of implementing a bio-film detector, and possibly disperser ("saboteur"), proved too ambitious to undertake over a limited project period of 10 weeks. It was determined, therefore, that the well-studied E-Coli biofilm would be investigated further, and that the Detector would in fact be applied to this biofilm platform. More detailed information concerning the previously investigated Biofilms can be found here: Future Extensions of Biofilm Detector.This is important to consider in future applications of the "Biofilm Detector".

Recently, a paper[1] was published describing the use of an engineered bacteriophage that would also produce DspB, an enzyme that breaks down one of the major structural components of biofilm. However, it was pointed out that bacteriophages target very specific cells, which may not be present in the biofilm under consideration. Furthermore, bacteria often evolve defences against phages. Thus, although the phage technique has proven that the principle of infecting a biofilm is a good strategy to disperse it, it is far from becoming usable outside the tightly controlled lab environments.

This project proposes an alternative method of biofilm dispersal. In short, a bacteria will be modified to express DspB as a response to three input signals - quorum sensing, biofilm presence, and anaerobic conditions. These bacteria can then be sprayed over a biofilm, which they infiltrate, and disperse.

There are three major assumptions that may prove problematic in this proposal:

1. That the saboteur bacteria will be able to penetrate the biofilm
2. That the saboteur bacteria will survive in the biofilm
3. That the quorum sensing mechanism will work

A list of references can be found at the bottom, that may be able to help addressing these concerns.

The Hrp system may be used with its three inputs: biofilm detection, quorum sensing, and anaerobic conditions sensing (inverted in V input). By inverting the anaerobic conditions sensing, a basal level (the 10% leakage of the V-inhibition) of DspB will be produced. As soon as anaerobic conditions are reached, V-expression is inhibited (the inversion) which kicks the Hrp system into full swing.

On Escherichia coli Bio-Films

E. coli is ubiquitous in human intestines, and is normally benign. However, a few strains can cause infections, of three types: urinary tract infection (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). Of these, urinary tract infection is the most common nosocomial infection, and is caused by E. coli in abou 80% of all cases. Further, the UTI infective strains are well documented[1].

Once again, creating a system that can detect the presence of the particular infectious strains would serve two useful purposes: one, it could help in the surveillance and prevention of e.coli infections. Two, and perhaps more importantly, it could serve as a proof of concept that synthetic biology can be used to produce cheap, reliable, and fast sensors for particular pathogens. Also, E.coli is usually used as an indicator for human fecal contamination of water.

One question is that, if e.coli is used as the chassis for the detection system, how would one avoid having the system activate itself? A method for separating the target e.coli from the system e.coli must be devised.