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 is optimum, in terms of the reponse time and the output fluorescence at the response time.

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

Preparation of reactions

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
   • Premix tube
   • S30 tubes
3. To prepare the commercial E.coli Cell Extract, carry out the following Procedure:
   
   1. First prepare a complete amino acid mixture for the extract solution: Add the 37.5µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 75µl. Each amino acid minus mixture is missing one type of amino acid.
   2. Take an eppendorf tube and add the 75µl of the E.coli complete amino acid mixture.
   3. Add 300µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   4. Then add 225µl of S30 Extract Circular too.
   5. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
4. Incubate the prepared cell extract mixture in the water bath set at 25°C.
5. Prepare the different DNA concentrations: ------
6. Put 51µl of each DNA concentration into an eppendorf tube and place them in the 25°C water bath.
7. Separate 20ul??? of maxipreped DNA of empty vector into an eppendorf tube and place in the water bath as well.
8. Prepare 45µl of 50nM solution of AHL for all the DNA concentrations:
   1. Aliquot 2.25µl of 1mM AHL into an eppendorf tube.
   2. Add in 42.75µl of water into the eppendorf to get the required dilution.
9. Put the eppendorf with the diluted AHL into the 25°C water bath too.

Loading Plate

1. Take the plate out of the incubation
2. Follow the schematic for the plate and begin by loading 40µl of the in vitro expression system into the correct wells.
3. Tap down the top of the plate to bring down any solution to bottom of the well.
4. Then to begin the reaction add 17µl of purified DNA sample to each well, as indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
5. Add 20µl of the empty vector into the three negative conrtol wells, as shown in the schematics.
6. Put in 3µl of AHL into each well, following the schematics.
7. Put 60µl of water into some empty wells in the middle of teh plates. These will be used to check the evaporation.
8. After the DNA and the AHL has been put into their respective wells, load the program on the PC to measure the fluorescence in the right wells.
9. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\ID\ OTR. The data from the fluoreometer will be exported here.
10. Each file should be named as the following:
    • construct-temp-time-date
11. While program loads, get the plate out of the water bath and wipe off the water on it.
12. Take a reading in the fluorometer. Before each measurement remember to tap down the solution and to remove the lid before placing in the fluorometer.
13. As soon as the reading has been taken, unload the plate and place the clear tape on the plate and place back in the incubator. Cover the plate with foil to prevent the DNA from getting bleached due to light. Make sure that the plate is not outside the water bath for longer than 5mins. Remember to close the plate holder of the fluorometer after each reading.
14. After an hour of incubation, load the program on the PC to measure the fluorescence in the right wells.
15. Take another fluorescence reading, repeating steps 10-13.
16. Take a reading every hour, until 6 hours have elapsed since the first reading.
17. After the last reading, measure the amount of water left in the wells (no DNA) to check the amount of fluid that has evaporated.
18. 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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