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| ==Biofilm detection using AHL as signals== | | ==Biofilm detection using AHL as signals== |
| http://aem.asm.org/cgi/reprint/67/2/575 | | http://aem.asm.org/cgi/reprint/67/2/575 |
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| ==Protocols for DNA concentration experiments==
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| 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.
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|
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| ===Aims===
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| *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.
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|
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| ===Equipment===
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| *Fluorometer + PC
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| *25°C water bath
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| *Fluorometer plate
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| *Gilson pipettes 200, 20, 10
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| *Eppendorf Tubes
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| *Stopwatch
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|
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| ===Reagents===
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| *[[IGEM:IMPERIAL/2007/Projects/Experimental Design/Protocol/Cell Extract |'''S30 E.coli Cell Extract''']]
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| *Nuclease Free water
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| *DNA
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|
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| ===Preparation of reactions===
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| #First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed.
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| #For the cell extract, get the following out of the cell extract kit:
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| #*A.A's from kits
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| #*Premix tube
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| #*S30 tubes
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| #To prepare the commercial E.coli Cell Extract, carry out the following Procedure:<br>
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| ##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.
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| ##Take an eppendorf tube and add the 75µl of the E.coli complete amino acid mixture.
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| ##Add 300µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
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| ##Then add 225µl of S30 Extract Circular too.
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| ##Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
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| #Incubate the prepared cell extract mixture in the water bath set at 25°C.
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| #Prepare the different DNA concentrations: ------
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| #Put 51µl of each DNA concentration into an eppendorf tube and place them in the 25°C water bath.
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| #Separate 20ul??? of maxipreped DNA of empty vector into an eppendorf tube and place in the water bath as well.
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| #Prepare 45µl of 50nM solution of AHL for all the DNA concentrations:
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| ##Aliquot 2.25µl of 1mM AHL into an eppendorf tube.
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| ##Add in 42.75µl of water into the eppendorf to get the required dilution.
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| #Put the eppendorf with the diluted AHL into the 25°C water bath too.
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|
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| ===Loading Plate===
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| #Take the plate out of the incubation
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| #Follow the schematic for the plate and begin by loading 40µl of the in vitro expression system into the correct wells.
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| #Tap down the top of the plate to bring down any solution to bottom of the well.
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| #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.
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| #Add 20µl of the empty vector into the three negative conrtol wells, as shown in the schematics.
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| #Then to start the reaction, put in 3µl of AHL into each well, following the schematics.
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| #Put 60µl of water into some empty wells in the middle of the plate. These will be used to check the evaporation.
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| #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.
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| #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.
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| #Each file with the reading should be named as the following:
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| #*construct-temp-time-date
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| #While program loads, get the plate out of the water bath and wipe off the water on it.
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| #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.
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| #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.
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| #After an hour of incubation, load the program on the PC to measure the fluorescence in the right wells.
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| #Take another fluorescence reading, repeating steps 10-13.
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| #Take a reading similarly every hour, until 6 hours have elapsed since the first reading.
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| #After the last reading, measure the amount of water left in the wells (no DNA) to check the amount of fluid that has evaporated.
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| #Wash off the plates with 70% ethanol and rinse with distilled water
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|
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| ===Schematic===
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|
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| {| border="1" cellpadding="1"
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| !<u>Well</u> || <u>Test Construct</u> !! <u> Concentration of DNA</u> !! <u>In vitro chassis</u>
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| |-
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| |<font color=blue>
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| |<font color=blue> pLux-luxR-pLux-GFP + AHL
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| |<font color=blue> Concetration 1
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| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 1
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 1
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>Empty vector + AHL (Negative control)
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>GFP (Positive conrtol)???
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue> pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 2
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 2
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 2
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>Empty vector + AHL (Negative control)
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>GFP (Positive conrtol)???
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue> pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 3
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 3
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>pLux-luxR-pLux-GFP + AHL
| |
| |<font color=blue> Concetration 3
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>Empty vector + AHL (Negative control)
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |<font color=blue>
| |
| |<font color=blue>GFP (Positive conrtol)???
| |
| |<font color=blue> -
| |
| |<font color=blue>Commercial E.coli extract
| |
| |-
| |
| |}
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|
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| <br=clear all>
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