IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Notes: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
 
(15 intermediate revisions by 3 users not shown)
Line 1: Line 1:
=Biofilm Detection: Notes=
=Infector Detector: Notes=
__NOTOC__
__NOTOC__


Line 8: Line 8:
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Design|Design]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Design|Design]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Modelling|Modelling]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Modelling|Modelling]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation|Implementation]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation|Fabrication]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/TestingValidation|Testing/Validation]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/TestingValidation|Testing]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Data Analysis|Data Analysis]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Validation|Validation]]</li>
<li id="current">[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Notes|Notes]]</li>
<li id="current">[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Notes|Notes]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/References|References]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/References|References]]</li>
Line 16: Line 18:
<br style="clear:both">
<br style="clear:both">


__TOC__
<!--__TOC__-->


==Making Biofilm==
*Freshly-grown growth-phase E. coli ZK1056 culture is added to sterile medium to a concentration of 5 × 107 cells/mL.
*Round 15 mm sterile glass coverslips are placed upright in the wells of a 24-well plate with sterile plastic stands
*Add 1 mL of E. coli solution to each well.
*The 24-well plate is immediately covered and transferred to a 30 °C incubator.


==AHL produced in biofilms:==
(Notes on the journal modeling AHL production in biofilms of the same bacterial population)
AHL synthesis is subject to autoinduction in which production of AHLs operates
as a positive feedback loop.
<br> Assumptions made in the model:
*All bacterial cells are physiologically identical with regard to size, shape and permeability of the cell membrane, as well as production and degradation rates of the signalling molecules
*Bacterial population exhibit a standard logistic growth pattern
*No metabolic or physiological lag is assumed
*At very low Cbc, the net rate of AHL production, h(Cbc), is assumed to be determined solely by the difference between basal production, Bp, and degradation of AHLs
* Degradation of AHLs is proportional to the concentration of AHL and occurs at a rate d*Cbc


Not considered in the model: permeability constant a, which is characteristic of the bacterial cell membrane, the diffusability of a given AHL, and the viscosity of the cell and the biofilm


Conclusions from the model: high concentrations of AHL inside cells could be achieved at very low population densities. Rapid rise in AHL concentration early in population growth, followed by a plateau, followed by another rise to a second plateau


== On Diffusion Through The Biofilm ==
==Biofilm detection using AHL as signals==
Effective diffusion depend on solute type and density of biofilm. Mean relative effective diffusive permeability (De/$aq) of different solutes:
http://aem.asm.org/cgi/reprint/67/2/575
*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
<br>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===
[[IMAGE:Biofilm.jpg|thumb|right|Biofilm Formation]]
There are three stages of biofilm formation:
# The initial attachments of microorganisms to a surface.
# After a certain density is reached a 'slime capsule' is built up. This capsule is composed of exopolysacchorides that are secreted from the cell.
#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]
 
==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
 
===Summary===
Our project is about detection of biofilms using the lux system. We will simulate the presence of biofilms by AHL producing E. coli. The sensitivity of the lux promoter under different conditions (concentration of AHL and temperature) will be assayed by a fluorescent reporter. This system will also be tested in vitro and in veso. Hrp system may be incorporated into the system to amplify the fluorescent output.

Latest revision as of 07:55, 26 September 2007

Infector Detector: Notes



Making Biofilm

  • Freshly-grown growth-phase E. coli ZK1056 culture is added to sterile medium to a concentration of 5 × 107 cells/mL.
  • Round 15 mm sterile glass coverslips are placed upright in the wells of a 24-well plate with sterile plastic stands
  • Add 1 mL of E. coli solution to each well.
  • The 24-well plate is immediately covered and transferred to a 30 °C incubator.

AHL produced in biofilms:

(Notes on the journal modeling AHL production in biofilms of the same bacterial population) AHL synthesis is subject to autoinduction in which production of AHLs operates as a positive feedback loop.
Assumptions made in the model:

  • All bacterial cells are physiologically identical with regard to size, shape and permeability of the cell membrane, as well as production and degradation rates of the signalling molecules
  • Bacterial population exhibit a standard logistic growth pattern
  • No metabolic or physiological lag is assumed
  • At very low Cbc, the net rate of AHL production, h(Cbc), is assumed to be determined solely by the difference between basal production, Bp, and degradation of AHLs
  • Degradation of AHLs is proportional to the concentration of AHL and occurs at a rate d*Cbc

Not considered in the model: permeability constant a, which is characteristic of the bacterial cell membrane, the diffusability of a given AHL, and the viscosity of the cell and the biofilm

Conclusions from the model: high concentrations of AHL inside cells could be achieved at very low population densities. Rapid rise in AHL concentration early in population growth, followed by a plateau, followed by another rise to a second plateau

Biofilm detection using AHL as signals

http://aem.asm.org/cgi/reprint/67/2/575

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:IMPERIAL/2007/Projects/Biofilm_Detector/Notes&oldid=154317"