User:Maira Tariq/sandbox

Application driven projects: (23/07/07)

• Detecting food spoiling
  • Detect pH change and temp change in surrounding environment
  • Visual change to show spoiling

1. Ternström A, Lindberg AM, and Molin G. Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus. J Appl Bacteriol. 1993 Jul;75(1):25-34. DOI:10.1111/j.1365-2672.1993.tb03403.x |

• Biofilm removal from surfaces, eg medical insrtuments, food etc.
  • Prosthetic hip joints: http://jcm.asm.org/cgi/reprint/37/10/3281 - but would need to replace
  • E coli biofilm: http://www.blackwell-synergy.com/links/doi/10.1046/j.1365-2958.2003.03490.x/full/?cookieSet=1
  • Using signalling within biofilms to destroy them: http://www.nature.com/nbt/journal/v21/n4/full/nbt0403-361.html
  • http://www.blackwell-synergy.com/links/doi/10.1046/j.1462-2920.2000.00128.x/abs/
  • http://iai.asm.org/cgi/reprint/IAI.01143-06v1.pdf
  • http://www.sciencemag.org/cgi/content/full/311/5764/1113
  • Catheter tubes

1. can use bacteria to detect biofilm on surface.
2. don't introduce bacteria inside as may accumulate
3. discard the catheter if biofilm detected

• Detect food for allergic substances eg nuts, dairy products etc.
• Indicate excess UV exposure of skin
• Biofilm to form lubricant(cartilage) on joints
  • Biocompatible
• Stimulate bacteria in a contolled way
  • Create 3D images
  • Biological-electronic interface
• Gut bacteria - assess nutritients in diet
• Layer of biofilm on stagnant water to block sunlight and oxygen
  • Kill mosquito cells
• HRP system applications:
  • Glucose/insulin regulation system
  • Problem: no receptor in E. coli to detect glucose directly

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

Protocols for DNA concentration experiments

Experiments to be carried out are to determine the optimum concentration of the pLux-luxR-pLux-GFP construct for the ID, in-vitro. Each concentration of DNA will be tested over a period of 6 hours, as it is expected that the system will respond within about 2-3 hours to AHL. The evaporation of the samples will be taken into account when analysing the data.

Aims

• To determine the concentration of DNA for which the response to AHL being induced optimum, in terms of the reponse time and the output fluorescence

Equipment

• Fluorometer + PC
• 25°C water bath
• Fluorometer plate
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes
• Stopwatch

Reagents

• S30 E.coli Cell Extract
• Nuclease Free water
• DNA

Protocols

1. First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed.
2. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • 2xPremix tubes (60ul each)
   • 2xS30 tubes (45ul each)
     
3. For Each DNA concentration Carry out the following Procedure:
   
4. Commercial E.coli Cell Extract:
   1. First prepare a complete amino acid mixture for both extract solutions: Add the 10µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 20µl. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench.
   2. Take an eppendorf tube and add 20µl of the E.coli complete amino acid mixture.
   3. Add 80µl of S30 Premix Without Amino Acid.
   4. Then add 60µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench.
5. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
6. Prepare the different DNA concentrations:
7. Separate 60µl of maxipreped DNA plasmid into an eppendorf tube and place them in their respective incubators as well.
8. Separate 20ul of maxipreped DNA of empty vector into separate tubes.
9. Fill some of the centre and side wells, which have no cell extract and DNA mixture in them, with 60ul of water. These samples will be used to measure the amount of evaporation of the samples.

Loading Plate

Day 1:

1. Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells.
2. Tap down the top of the lid to bring down any solution to bottom of the well.
3. Remove cover off the 96 well plate and place in the fluorometer.
4. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\CBD\ OTR. The data from the fluoreometer will be exported here. Each file should be named as the following:
   • construct-temp-time-date
5. Take a fluorometer reading. This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
6. Then to begin the reaction add 20µl of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
7. Place cover back on and place back in the respective incubators. Cover the plates with foil to prevent the DNA from getting bleached due to light.
8. After 30 minutes of incubation measure the fluorescence by repeating procedure 3-4 above.
9. Keep taking measurments like this every 30 mins, until 6 hours have elapsed since the first reading, with DNA. Before each measurement be careful to remember to tap down the solution and to remove the lid before placing in the fluorometer.
10. After the last reading, check the samples under a UV light to see if the fluorescence reached is visible or not. Turn the lights off in the room before proceeding with this.
11. Then measure the amount of water left in the wells (no DNA) to check the amount of fluid that has evaporated.
12. Leave the plates incubated in 4°C over a period of two nights, with the cover and the foil on. This will allow to test for the lifespan of the DNA and cell extract mixture, after it has reacted (see application specs).
    

Day 4:

1. Take a last fluorometer reading of each plate
2. Measure the amount of samples left in the wells.
3. Wash off the plates with 70% ethanol and rinse with distilled water

Schematic

Well	Test Construct	In vitro chassis	Vol of in vitro chassis (ul)
	pTet	Commercial E.coli extract
	pTet	Commercial E.coli extract
	pTet	Commercial E.coli extract
	pTet	Commercial E.coli extract
	pT7	Commercial E.coli extract
	pT7	Commercial E.coli extract
	pT7	Commercial E.coli extract
	pT7	Commercial E.coli extract

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