Team:Wisconsin-Madison/experiments

From 2010.igem.org

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<html>
 
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<ol>
 
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<li> Measure OD600
 
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<li> To inactivate EPS-degrading enzymes and completely release EPS from cell surface:
 
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<ul>
 
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<li> Boil sample for 15 min
 
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<li> Cool to room temp
 
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<li> Centrifuge at 14,000g for 30 min at 4°C
 
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</ul>
 
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<li> # Add three volumes of ethanol to 40 ml of supernatant fraction
 
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<li> # Place in 4°C overnight
 
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<li> # Centrifuge at 14,000g for 30 min at 4°C
 
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<li># Dissolve pellet in 1 ml of sterile distilled water
 
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# Quantifications: Use negative controls of glucose and sterile distilled water
 
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## Add 100 ul of colonic acid preparation to 1 ml of sterile distilled water
 
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## Mix 4.5 ml of H2SO4/H2O (6:1 v/v)
 
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## Heat mixture to 100°C for 20 min
 
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## Cool to room temperature
 
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## Measure absorbance at 396 nm and 427 nm
 
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## Add 100 μL of cysteine hydrochloride
 
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## Measure absorbance at 396 nm and 427 nm
 
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## Difference in these measurements (after subtracted from pre-cysteine addition absorbance) can be directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 μg/ml to 100 μg/ml
 
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</html>
 
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See entire procedure : Download .pdf
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See entire procedure : [[Media:UW_colonicacidassay.pdf Download]]
See original reference: [[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link]]
See original reference: [[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link]]

Revision as of 01:51, 20 October 2010

Contents

Enzyme Treatment

Encapsulation

Colonic Acid Quantification

Parts used in this experiment
Part Number Function Expression Type Zip File
<partinfo>BBa_k318500</partinfo> Produces Trascription Factor RcsB Inducible - IPTG 500
<partinfo>BBa_k318501</partinfo> Produces Trascription Factor RcsA Inducible - IPTG 501
<partinfo>BBa_k318502</partinfo> Produces Trascription Factor RcsA & RcsB Inducible - IPTG 502 <partinfo>BBa_k200021</partinfo> Empty vector - control Inducible - IPTG



See entire procedure : Media:UW_colonicacidassay.pdf Download See original reference: http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link

Cell Survivability Testing

Parts used in this experiment
Part Number Function Expression Type Zip File
<partinfo>BBa_k318500</partinfo> Produces Trascription Factor RcsB Inducible - IPTG 500
<partinfo>BBa_k318501</partinfo> Produces Trascription Factor RcsA Inducible - IPTG 501
<partinfo>BBa_k318502</partinfo> Produces Trascription Factor RcsA & RcsB Inducible - IPTG 502

Best Combination


Inducible-Repressible Expression

Characterize pH Promoters

Parts Used: <partinfo>BBa_k318513</partinfo>

Amount of Regulators

IR-System - Arabinose

Parts Used: <partinfo>BBa_k318509</partinfo>, <partinfo>BBa_k318510</partinfo>, <partinfo>BBa_K318511</partinfo>, <partinfo>BBa_K318506</partinfo>

IR-System - pH

Parts Used: <partinfo>BBa_TUNED Part</partinfo>, <partinfo>BBa_K318506</partinfo>

IR-Lysis - pH

Parts Used: <partinfo>BBa_TUNED Part</partinfo>, <partinfo>BBa_K318507</partinfo>

Bile Induction

<partinfo>BBa_K318516</partinfo>, <partinfo>BBa_Lysis Part</partinfo> <partinfo>BBa_K318516</partinfo>, <partinfo>BBa_LYSIS Part</partinfo>

Encryption

Laboratory Notebooks

Media:Wisconsin-Madison2010_Notebook1.pdf

Media:Wisconsin-Madison2010_Notebook2.pdf

Media:Wisconsin-Madison2010_Notebook3.pdf

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