IGEM:IMPERIAL/2006/Protocols/Biosensortest: Difference between revisions

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''Materials:''
''Materials:''
*Enzyme Acylase from Porcine Kidney [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A3010 (more info: sigmaaldrich)], stored as powder in freezer
*Enzyme Acylase from Porcine Kidney [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A3010 (more info: sigmaaldrich)], stored as powder in freezer
*AHL (N-(β-Ketocaproyl)-DL-homoserine lactone) [http://www.sigmaaldrich.com/catalog/search/ProductDetail?ProdNo=K3255&Brand=SIGMA (more info & catalogue no: sigmaaldrich)], comes as powder to be stored in -20{{c}} freezer
: - has been diluted into different stock concentrations (the solutions are stored in the -20{{c}} freezer)
*(Potassium Phosphate (KPO<sub>4</sub>) buffer)
*(Potassium Phosphate (KPO<sub>4</sub>) buffer)
*MiliQ
*MiliQ
*(LB Medium)
*LB Medium
*T9002 assay being cultured up following the [http://openwetware.org/wiki/IGEM:IMPERIAL/Protocols/T9002 T9002 testing protocol]
*T9002 assay being cultured up following the [http://openwetware.org/wiki/IGEM:IMPERIAL/Protocols/T9002 T9002 testing protocol]


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*Before starting, set up a 25{{c}} waterbath (Dr. O'Hare's Lab?)
*Before starting, set up a 25{{c}} waterbath
*Prepare the following solutions by adding Columns 1,3 and 6 ON ICE. Add the enzyme at last:  
1.) Prepare the solutions as outlined in the table by adding Columns 1,3 and 6 on ice. Add the enzyme at last.
::NOTE: For an initial test, only test rows D, F, G
2.)  Again, prepare the solutions as outlined in the table. However, instead of using LB in Coloumn 1, use MiliQ. Add Columns 1 (MiliQ),3 and 6 on ice; add the enzyme at last.
 
 
*REMARK: Because of the size of the pH glass electrode, the volumes used (see table below) had to be at least 1.5mL. If a smaller pH electrode is used, smaller volumes can be used to be tested.


{| border="3" cellpadding="3"
{| border="3" cellpadding="3"
!<u>Label</u>||(1)<br><u>LB medium ({{uL}})</u>||(2)<br><u>Available Stock Concentr of AHL</u> !! (3)<br><u>Volume of AHL to add ({{uL}})</u> !! (4)<br><u>Final AHL Concentration</u> !!(5)<br><u>Amount of AHL present <br>in solution</u>!!(6)<br><u>Volume of Enzyme to add</u>
!<u>Label</u>||(1)<br><u>LB medium ({{uL}})</u>||(2)<br><u>Available Stock Concentr<br>of AHL</u> !! (3)<br><u>Volume of AHL to add ({{uL}})</u> !! (4)<br><u>Final AHL Concentration</u> !!(5)<br><u>Amount of AHL present <br>in solution</u>!!(6)<br><u>Volume of Enzyme to add</u>
|-
|-
!A
!A
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|-
|-
!D
!D
|450
|1800
|1000uM
|200
|100uM
|400nm
|16uL of Soln 3 (=0.8units)
|-
!Control for D
|1800
|1000uM
|1000uM
|50
|200
|100uM
|100uM
|100nm
|400nm
|4uL of Soln 3 (=0.8units)
|N/A
|-
|-
!E
!E
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|-
|-
!F
!F
|990
|1980
|1000uM
|20
|10uM
|20nm
|8uL of Soln 5 (=0.08units)
|-
!Control for F
|1980
|1000uM
|1000uM
|10
|20
|10uM
|10uM
|10nm
|20nm
|4uL of Soln 5 (=0.08units)
|N/A
|-
|-
!G
!G
|990
|1980
|100uM
|100uM
|10
|20
|1uM
|1uM
|1nm
|2nm
|4uL of Soln 6 (=0.008units)
|8uL of Soln 6 (=0.008units)
|-
!Control for G
|1980
|100uM
|20
|1uM
|2nm
|N/A
|-
|-
!H
!H
|990
|1980
|10uM
|10uM
|10
|20
|100nM
|100nM
|0.1nm
|0.2nm
|4uL of Soln 7 (=8E-4units)
|8uL of Soln 7 (=8E-4units)
|-
|-
!I
!I
|990
|1980
|5uM
|5uM
|10
|20
|100nM
|50nM
|0.1nm
|0.2nm
|4uL of Soln 8 (=8E-5units)
|8uL of Soln 8 (=8E-5units)
|-
|-
!J
!J
|990
|1980
|1uM
|1uM
|10
|20
|10nM
|10nM
|0.01nm
|0.02nm
|4uL of Soln 9 (=8E-6units)
|8uL of Soln 9 (=8E-6units)
|-
|-
!K
!K
|990
|1980
|500nM
|500nM
|10
|20
|5nM
|5nM
|0.005nm
|0.01nm
|4uL of Soln 10 (=4E-6units)
|8uL of Soln 10 (=4E-6units)
|-
|-
!L
!L
|990
|1980
|100nM
|100nM
|10
|20
|1nM
|1nM
|0.001nm
|0.002nm
|4uL of Soln 11 (=8E-7units)
|8uL of Soln 11 (=8E-7units)
|-
|-
!M
!M
|990
|1980
|50nM
|50nM
|10
|20
|0.5nM
|0.5nM
|0.0005nm
|0.001nm
|4uL of Soln 12 (=4E-7units)
|8uL of Soln 12 (=4E-7units)
|-
|-
!N
!N
|990
|1980
|10nM
|10nM
|10
|20
|0.1nM
|0.1nM
|0.00005nm
|0.0001nm
|4uL of Soln 13 (=8E-8units)
|8uL of Soln 13 (=8E-8units)
|-
|-
!O
!O
|990
|1980
|1nM
|1nM
|10
|20
|0.01nM
|0.01nM
|0.00001nm
|0.00002nm
|4uL of Soln 14 (=8E-9units)
|8uL of Soln 14 (=8E-9units)
|-
|-
!P
!Control LB
|1000
|2000
|N/A
|N/A
|N/A
|N/A
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|N/A
|N/A
|N/A
|N/A
|-
!Control Enzyme
|2000
|N/A
|N/A
|N/A
|N/A
|16uL of Soln 3 (=0.8units)
|}
|}
(Note: The seemingly arbitrary numbers in row A-E were chosen because of the limited stock and concentrations of AHL available)
(Note: The seemingly arbitrary numbers in row A-E were chosen because of the limited stock and concentrations of AHL available)
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If activity of the enzyme has to be checked:  
If activity of the enzyme has to be checked:  
*Follow the [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols/BiosensorEnzymeAct Biosensor Testing: Enzyme Activity protocol] afterwards
*Follow the [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols/BiosensorEnzymeAct Biosensor Testing: Enzyme Activity protocol] afterwards
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Latest revision as of 09:49, 30 October 2006

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Biosensor: Calibration & Characterization of the Sensitivity of the Sensor

Motivation

In order to use the biosensor for later experiments, a calibration curve has to be established first. With this testing protocol, the relation between pH and AHL concentration should be extracted. Thus, the sensitivity of the sensor can be determined and characterized.

  • JS: is it possible that we can get a continuous readout of the pH, so we can assess the variability in the pH meter over time? Depending upon if it hits a spot of a higher concentration of AHL, it might increase the pH level (or decrease) if it is not stirred properly.
  • CS: Yes, the pH will be read in real-time to get an idea of the activity of the enzyme (that is why everything is prepared on ice first). During digest in the 25[[:Category:{{{1}}}|{{{1}}}]]waterbath, the pH should be observed for any changes in time until it reaches a steady value. The whole solution should have been well-mixed before.

Equipment and Materials

Equipment:

  • pH meter
  • Eppendorf Tubes
  • Small White Cap Tubes
  • Gilson Pippettes

Materials:

- has been diluted into different stock concentrations (the solutions are stored in the -20[[:Category:{{{1}}}|{{{1}}}]] freezer)
  • (Potassium Phosphate (KPO4) buffer)
  • MiliQ
  • LB Medium
  • T9002 assay being cultured up following the T9002 testing protocol

Protocol

  • Dissolving the enzyme: (only need to do this the very first time, or if you run out of enzyme mix)
Soln 1: Dissolve 1mg enzyme in 1mL KPO4 buffer (-> concentration of 2000units/mL)
Soln 2: Take 0.5mL of Soln 1 and dilute in 0.5mL MiliQ (-> concentration of 1000units/mL)
Soln 3: Take 0.1mL of Soln 1 and dilute in 0.9mL MiliQ (-> concentration of 200units/mL)
Soln 4: Take 0.1mL of Soln 2 and dilute in 0.9mL MiliQ (-> concentration of 100units/mL)
Soln 5: Take 0.1mL of Soln 3 and dilute in 0.9mL MiliQ (-> concentration of 20units/mL)
Soln 6: Take 0.1mL of Soln 5 and dilute in 0.9mL MiliQ (-> concentration of 2units/mL)
Soln 7: Take 0.1mL of Soln 6 and dilute in 0.9mL MiliQ (-> concentration of 0.2units/mL)
Soln 8: Take 0.1mL of Soln 7 and dilute in 0.9mL MiliQ (-> concentration of 0.02units/mL)
Soln 9: Take 0.15mL of Soln 8 and dilute in 1.35mL MiliQ (-> concentration of 0.002units/mL)
Soln 10: Take 0.5mL of Soln 9 and dilute in 0.5mL MiliQ (-> concentration of 0.001units/mL)
Soln 11: Take 0.1mL of Soln 10 and dilute in 0.9mL MiliQ (-> concentration of 0.0002units/mL)
Soln 12: Take 0.1mL of Soln 10 and dilute in 0.9mL MiliQ (-> concentration of 0.0001units/mL)
Soln 13: Take 0.1mL of Soln 11 and dilute in 0.9mL MiliQ (-> concentration of 0.00002units/mL) 
Soln 14: Take 0.1mL of Soln 13 and dilute in 0.9mL MiliQ (-> concentration of 0.000002units/mL)


  • Before starting, set up a 25[[:Category:{{{1}}}|{{{1}}}]] waterbath

1.) Prepare the solutions as outlined in the table by adding Columns 1,3 and 6 on ice. Add the enzyme at last.

NOTE: For an initial test, only test rows D, F, G

2.) Again, prepare the solutions as outlined in the table. However, instead of using LB in Coloumn 1, use MiliQ. Add Columns 1 (MiliQ),3 and 6 on ice; add the enzyme at last.


  • REMARK: Because of the size of the pH glass electrode, the volumes used (see table below) had to be at least 1.5mL. If a smaller pH electrode is used, smaller volumes can be used to be tested.
Label (1)
LB medium (μL)		 (2)
Available Stock Concentr
of AHL		 (3)
Volume of AHL to add (μL)		 (4)
Final AHL Concentration		 (5)
Amount of AHL present
in solution		 (6)
Volume of Enzyme to add
A 0 1000uM 200 1000uM 1um 4uL of Soln 1 (=8units)
B 100 1000uM 100 500uM 500nm 4uL of Soln 2 (=4units)
C 0 100uM 200 100uM 100nm 4uL of Soln 3 (=0.8units)
D 1800 1000uM 200 100uM 400nm 16uL of Soln 3 (=0.8units)
Control for D 1800 1000uM 200 100uM 400nm N/A
E 100 100uM 100 50uM 50nm 4uL of Soln 4 (=0.4units)
F 1980 1000uM 20 10uM 20nm 8uL of Soln 5 (=0.08units)
Control for F 1980 1000uM 20 10uM 20nm N/A
G 1980 100uM 20 1uM 2nm 8uL of Soln 6 (=0.008units)
Control for G 1980 100uM 20 1uM 2nm N/A
H 1980 10uM 20 100nM 0.2nm 8uL of Soln 7 (=8E-4units)
I 1980 5uM 20 50nM 0.2nm 8uL of Soln 8 (=8E-5units)
J 1980 1uM 20 10nM 0.02nm 8uL of Soln 9 (=8E-6units)
K 1980 500nM 20 5nM 0.01nm 8uL of Soln 10 (=4E-6units)
L 1980 100nM 20 1nM 0.002nm 8uL of Soln 11 (=8E-7units)
M 1980 50nM 20 0.5nM 0.001nm 8uL of Soln 12 (=4E-7units)
N 1980 10nM 20 0.1nM 0.0001nm 8uL of Soln 13 (=8E-8units)
O 1980 1nM 20 0.01nM 0.00002nm 8uL of Soln 14 (=8E-9units)
Control LB 2000 N/A N/A N/A N/A N/A
Control Enzyme 2000 N/A N/A N/A N/A 16uL of Soln 3 (=0.8units)

(Note: The seemingly arbitrary numbers in row A-E were chosen because of the limited stock and concentrations of AHL available)

  • Add the enzyme just before putting the mixtures into 25[[:Category:{{{1}}}|{{{1}}}]] waterbath
  • Vortex solutions shortly to mix the content
  • Measure and write down the pH of all solutions before taking them off ice
  • Make a note of the time and take the solutions off the ice into a 25[[:Category:{{{1}}}|{{{1}}}]] waterbath (make sure the contents is well-mixed before)
  • Immediately after putting the solutions into the waterbath - stick in the pH electrode to get a real-time reading
    • Note down the initial pH of all solutions
    • Observe and write down all changes in pH over the next 10min. (take a reading every minute)
    • After 10min. the pH should have reached a steady value, if not, leave until it does not change any more
    • Write down the final pH


If activity of the enzyme has to be checked:

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