Team:Wisconsin-Madison/experiments

From 2010.igem.org

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====Quantification of Colonic Acid Production====
====Quantification of Colonic Acid Production====
'''Parts Used:''' <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>
'''Parts Used:''' <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>
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# Prepare overnight liquid culture of 50 ml sample
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# Measure OD600
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# To inactivate EPS-degrading enzymes and completely release EPS from cell surface:
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## Boil sample for 15 min
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## Cool to room temp
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## Centrifuge at 14,000g for 30 min at 4°C
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# Add three volumes of ethanol to 40 ml of supernatant fraction
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# Place in 4°C overnight
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# Centrifuge at 14,000g for 30 min at 4°C
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# 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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See entire procedure : Download .pdf
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See original reference: [[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link]]
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====Cell Survivability Testing====
====Cell Survivability Testing====
'''Parts Used:''' <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>
'''Parts Used:''' <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>

Revision as of 00:34, 28 September 2010

Contents

Enzyme Treatment

Encapsulation

Quantification of Colonic Acid Production

Parts Used: <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>

  1. Prepare overnight liquid culture of 50 ml sample
  2. Measure OD600
  3. To inactivate EPS-degrading enzymes and completely release EPS from cell surface:
    1. Boil sample for 15 min
    2. Cool to room temp
    3. Centrifuge at 14,000g for 30 min at 4°C
  4. Add three volumes of ethanol to 40 ml of supernatant fraction
  5. Place in 4°C overnight
  6. Centrifuge at 14,000g for 30 min at 4°C
  7. Dissolve pellet in 1 ml of sterile distilled water
  8. Quantifications: Use negative controls of glucose and sterile distilled water
    1. Add 100 ul of colonic acid preparation to 1 ml of sterile distilled water
    2. Mix 4.5 ml of H2SO4/H2O (6:1 v/v)
    3. Heat mixture to 100°C for 20 min
    4. Cool to room temperature
    5. Measure absorbance at 396 nm and 427 nm
    6. Add 100 μL of cysteine hydrochloride
    7. Measure absorbance at 396 nm and 427 nm
    8. 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


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

Cell Survivability Testing

Parts Used: <partinfo>BBa_k318500</partinfo>, <partinfo>BBa_k318501</partinfo>, <partinfo>BBa_K318503</partinfo>

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

Experimental Notebooks

Media:Wisconsin-Madison2010_Notebook1.pdf

Media:Wisconsin-Madison2010_Notebook2.pdf

Media:Wisconsin-Madison2010_Notebook3.pdf

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