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> [http://www.nature.com/ismej/journal/v2/n6/full/ismej200824a.html paper]
 
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
 
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>
 
<u>Colonic acid assay </u>
 
This method is based on the specific difference in absorbance at 396 and 427 nm after reacting fucose with sulfuric acid and cysteine hydrochloride
 
1. Measure the fucose concentration to determine colonic acid concentration
 
2. Use a L-fucose (Acros Organics) calibration curve from 10–60  g ml- 1
 
3. normalise these values by cell turbidity at 600 nm
 
4. For negative control, glucose was assayed. It should not produce a significant signal.
 
 
<b>Note: Perform all experiments with two independent cultures.</b>
 
 
Another assay for colonic acid from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-4MMWHP9-1&_user=217827&_coverDate=03%2F16%2F2007&_rdoc=1&_fmt=full&_orig=search&_cdi=6899&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=98bee864d6a8931008db26c024e105e0#secx10 here]
 
Principle : Measure fucose concentration
 
 
1. Dilute 10 to 100 μl of the colanic acid preparation to 1 ml with distilled water
 
 
2. mixed it with 4.5 ml of H2SO4/H2O (6:1; v/v) at room temperature
 
 
3. heat at 100 °C for 20 min
 
 
4. cooled it  down to room temperature
 
 
5. For each sample measure absorbance at 396 nm and 427 nm was measured
 
 
a. directly (control sample (A-co))
 
 
b. After addition of 100 μl of cysteine hydrochloride (cysteine sample (A-cy)).
 
 
6. Heat with H2SO4 to yield brown products absorbing between 396 nm and 427 nm.
 
 
7. Subtract the absorption due to this unspecific reaction from the total absorption of the sample: A396-co and A427-co were subtracted from A396-cy and A427-cy, respectively, to obtain ΔA396 and ΔA427.
 
 
8. Values of (ΔA396–ΔA427) were directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml.
 
 
===Trehalose===


[http://www.biokits.com/moreinfos.html?id=1266 Sigma's glucose test kit]
==Module 3==
[[IGEM:IMPERIAL/2009/Assays_Protocols/M3_heatinduction |Heat induction Assays]]


Reference [http://www.pnas.org/content/99/15/9727.full paper]
[[IGEM:IMPERIAL/2009/Assays_Protocols/M3_Assays |Cell Death Assays]]


1. Extract trehalose by boiling cell pellets at 95degrees for 20mins.
=Cloning Strategies=


2. Convert trehalose in the supernatant to glucose with trehalase obtained from sigma.
[[IGEM:IMPERIAL/2009/Assays_Protocols/Cloning_Strategy_M1&T | Cloning Strategy for Module 1 and Timer]]


3. Measured this by a [http://www.biokits.com/moreinfos.html?id=1266 glucose assay kit from sigma].
[[IGEM:IMPERIAL/2009/Assays_Protocols/Cloning_strategy_M2 | Cloning Strategy for Module 2]]


4. Determine the preexisting glucose in each sample was determined in a control reaction without trehalase


5. Subtracted this from total glucose
==Testing Constructs==




alternatively, use [http://www.biokits.com/moreinfos.html?id=360 sigma's trehalose test kit]
[[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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