Team:Wisconsin-Madison/experiments

From 2010.igem.org

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## Measure absorbance at 396 nm and 427 nm
## Measure absorbance at 396 nm and 427 nm
## 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
## 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
See entire procedure : Download .pdf

Revision as of 20:49, 30 September 2010

Enzyme Treatment

Encapsulation

Quantification of Colonic Acid Production

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

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