User:Maira Tariq/sandbox

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.

The concentrations of DNA that will be tested are: 1, 2, 4, 6 and 10µg. 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 x 7
• Stopwatch
• Foil
• Clear tape

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. Place the 96well plate into the 25°C water bath
3. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • Premix tube
   • S30 tubes
4. 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 30µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 60µl. Each amino acid minus mixture is missing one type of amino acid.
   2. Take an eppendorf tube and add the 60µl of the E.coli complete amino acid mixture.
   3. Add 240µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   4. Then add 180µl of S30 Extract Circular too.
   5. This mixture is for all the samples. Label the tube.
   6. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
5. Incubate the prepared cell extract mixtures in the water bath set at 25°C.
6. Prepare a 36µl of 50nM solution of AHL for all the DNA concentrations:
   1. Aliquot 1.8µl of 1mM AHL into an eppendorf tube.
   2. Add in 34.2µl of water into the eppendorf to get the required dilution and label it.
7. Add the diluted AHL into the eppendorf tube with the cell extract and place back in the water bath.
8. Prepare the different DNA concentrations: 1µg, 2µg, 4µg, 6µg and 10µg. (ask about how to get different []s)
9. Put 34µl of each DNA concentration into a seperate, labeled eppendorf tube and place them in the 25°C water bath.

Loading Plate

1. Take the plate out of the incubation.
2. Follow the schematic for the plate and begin by loading 43µl of the in vitro expression system with AHL into each of the wells.
3. Tap down the top of the plate to bring down any solution to bottom of the well.
4. Then 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 17µl of distilled water into the two negative conrtol wells, as shown in the schematics.
6. Put 60µl of water into some empty wells in the middle of the plate. These will be used to check for evaporation.
7. After the DNA and the cell extract mixtures have been put into their respective wells, load the program on the PC to measure the fluorescence in the right wells.
8. 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.
9. Each file with the reading should be named as the following:
   • construct-temp-time-date
10. While the program loads, get the plate out of the water bath and wipe off the water on it.
11. Take a reading in the fluorometer. Before each measurement remember to tap down the solution and to remove the clear tape on it before placing in the fluorometer.
12. As soon as the reading has been taken, unload the plate and place the clear tape on the plate and place back in the water bath. 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.
13. After 30 mins of incubation, load the program on the PC again, to measure the fluorescence in the right wells.
14. Take another fluorescence reading, repeating steps 9-13.
15. Take a reading similarly every half an hour, until 6 hours have elapsed since the first reading.
16. After the last reading, measure the amount of water left in the wells (with no cell extract mixture) to check the amount of fluid that has evaporated.
17. Wash off the plates with 70% ethanol and rinse with distilled water

Schematic

Well	Test Construct	Concentration of DNA	In vitro chassis
	Distilled Water + AHL (Negative control)	0mg	Commercial E.coli extract
	Distilled Water + AHL (Negative control)	0mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	1mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	1mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	2mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	2mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	4mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	4mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	6mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	6mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	10mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	10mg	Commercial E.coli extract

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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