IGEM:IMPERIAL/2009/Assays Protocols: Difference between revisions

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=Current Wet Lab Plan=
[[IGEM:IMPERIAL/2009/Assays_Protocols/WetLab_Plan | Step By Step Wet-Lab Plan]]
= Assays =  
= Assays =  
==Functional Assays==
<b>Capsule: Is it able to withstand Stomach pH and protease conditions? </b>
We must test the functionality of the complete capsule, to see if it can withstand the conditions present in the stomach.
This can be done experimentally by recreating the stomach conditions in the lab, and seeing the effects on the bacteria. This could include quantifying the number of bacteria broken down.
<b>Trehalose Functionality: Does it help protect against dessication? </b>
As well as testing for the presence of trehalose, it may be useful to test to see if it performs its required function within our cells ie to protect against dessicating conditions.
* As trehalose is naturally present in E.coli, is it ok to assume that its presence will protect against dessication?
<br>
==Shopping Lists!==
[[IGEM:IMPERIAL/2009/Assays_Protocols/Shopping | Shopping]]


==Module 1==
==Module 1==
=== Cellulase ===
=== Cellulase ===
Cellulase Assay from Sigma Aldrich <br>
Cellulase Assay from Sigma Aldrich <br>
[[Media:Cellulase Assay.pdf | Cellulase Assay (Sigma)]]
[[Media:Cellulase Assay.pdf | Cellulase Assay (Sigma)]] &nbsp; <b>Quantitative</b>
<br><br>
<br><br>
Alternative Cellulase Assay <br>
[http://www.worthington-biochem.com/CEL/assay.html | Cellulase Assay]


<i>Feedback - In vitro or vivo? Do we need to purify enzyme for assay? presumably so, as requires measurement of glucose concentation, therefore not good to use cell extract </i>
<br>
<br>


=== Phenylalanine Hydroxylase (PAH)===
=== Phenylalanine Hydroxylase (PAH)===
Phenylalanine Hydroxylase Assay from Sigma Aldrich : <br>
Phenylalanine Hydroxylase Assay from Sigma Aldrich : <br>
[[Media:PAH Assay.pdf | PAH Assay]]
[[Media:PAH Assay.pdf | PAH Assay]] &nbsp; <b>Quantitative</b>
 


==Module 2==
==Module 2==
[[IGEM:IMPERIAL/2009/Assays_Protocols/Colanic_Acid | Colanic Acid Assay]]


===Colonic acid===
[[IGEM:IMPERIAL/2009/Assays_Protocols/Trehalose | Trehalose Assay]]
 
<br><br>
<u>Quantification of total EPS</u>
 
1. collect 60 mg of mucoid or non-mucoid bacterial culture from the surface of LB agar plates incubating for 12-h at 37 °C
 
2. resuspended in 1 ml sterile distilled water by vortex
 
3. determine cell concentration by cell turbidity at 600 nm
 
4. To inactivate EPS-degrading enzymes and release EPS
 
a. boil resuspended culture for 10 min
b. cool to room temperature
c. centrifuge at16000g for 10min
d. save supernatant fraction at - 20 °C for quantification
 
5. use the anthrone-H2SO4 assay to quantify total EPS Mendrygal and González, 2000
a. prepare 0.4 ml suitably-diluted sample
b. mixed it with 0.1 ml of fresh 2% anthrone in ethyl acetate in a 10 ml glass test tube.
c. Add 1ml of 95–97% H2SO4 into each sample
d. cooled for 10 min to room temperature
e. determine the value of the absorbance at 620 nm
f. Glucose equivalents in the EPS samples were quantified using a calibration curve with glucose from 10 to 80  g ml- 1
g. Normalise these values by cell turbidity at 600 nm


<b>Note: Perform all experiments with two independent cultures.</b>
==Module 3==
[[IGEM:IMPERIAL/2009/Assays_Protocols/M3_heatinduction |Heat induction Assays]]


<u>Colonic acid assay </u>
[[IGEM:IMPERIAL/2009/Assays_Protocols/M3_Assays |Cell Death Assays]]


This method is based on the specific difference in absorbance at 396 and 427 nm after reacting fucose with sulfuric acid and cysteine hydrochloride
=Cloning Strategies=


1. Measure the fucose concentration to determine colonic acid concentration
[[IGEM:IMPERIAL/2009/Assays_Protocols/Cloning_Strategy_M1&T | Cloning Strategy for Module 1 and Timer]]


2. Use a L-fucose (Acros Organics) calibration curve from 10–60  g ml- 1
[[IGEM:IMPERIAL/2009/Assays_Protocols/Cloning_strategy_M2 | Cloning Strategy for Module 2]]


3. normalise these values by cell turbidity at 600 nm


4. For negative control, glucose was assayed. It should not produce a significant signal.
==Testing Constructs==




<b>Note: Perform all experiments with two independent cultures.</b>
[[IGEM:IMPERIAL/2009/Assays_Protocols/Testing_Constructs| Testing Constructs for all modules]]

Latest revision as of 06:46, 7 August 2009

Current Wet Lab Plan

Step By Step Wet-Lab Plan


Assays

Functional Assays

Capsule: Is it able to withstand Stomach pH and protease conditions? We must test the functionality of the complete capsule, to see if it can withstand the conditions present in the stomach. This can be done experimentally by recreating the stomach conditions in the lab, and seeing the effects on the bacteria. This could include quantifying the number of bacteria broken down.

Trehalose Functionality: Does it help protect against dessication? As well as testing for the presence of trehalose, it may be useful to test to see if it performs its required function within our cells ie to protect against dessicating conditions.

  • As trehalose is naturally present in E.coli, is it ok to assume that its presence will protect against dessication?


Shopping Lists!

Shopping

Module 1

Cellulase

Cellulase Assay from Sigma Aldrich
Cellulase Assay (Sigma)   Quantitative


Feedback - In vitro or vivo? Do we need to purify enzyme for assay? presumably so, as requires measurement of glucose concentation, therefore not good to use cell extract

Phenylalanine Hydroxylase (PAH)

Phenylalanine Hydroxylase Assay from Sigma Aldrich :
PAH Assay   Quantitative

Module 2

Colanic Acid Assay

Trehalose Assay

Module 3

Heat induction Assays

Cell Death Assays

Cloning Strategies

Cloning Strategy for Module 1 and Timer

Cloning Strategy for Module 2


Testing Constructs

Testing Constructs for all modules

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