Team:Wisconsin-Madison/experiments

Enzyme Treatment

Encapsulation

Colonic Acid Quantification

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

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