Team:Wisconsin-Madison/experiments

From 2010.igem.org

Enzyme Treatment

Encapsulation

Quantification of Colonic Acid Production

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==== {| border="1" cellpadding="5" cellspacing="0" align="center" |+ align="center" style="color:#000000;" |''Parts used in this experiment'' |- |'''Part Number''' |'''Function''' |'''Expression Type''' |'''Zip File''' |- |BBa_k318500 |Produces Trascription Factor RcsB |Inducible - IPTG |500 |- |BBa_k318501 |Produces Trascription Factor RcsA |Inducible - IPTG |501 |- |BBa_k318502 |Produces Trascription Factor RcsA & RcsB |Inducible - IPTG |502 |} ====Best Combination====
   ===Inducible-Repressible Expression=== ====Characterize pH Promoters==== '''Parts Used:''' BBa_k318513 ====Amount of Regulators==== ====IR-System - Arabinose==== '''Parts Used:''' BBa_k318509, BBa_k318510, BBa_K318511, BBa_K318506 ====IR-System - pH==== '''Parts Used:''' BBa_TUNED Part, BBa_K318506 ====IR-Lysis - pH==== '''Parts Used:''' BBa_TUNED Part, BBa_K318507
   ===Bile Induction=== BBa_K318516, BBa_Lysis Part BBa_K318516, BBa_LYSIS Part
   ==Encryption== ===Laboratory Notebooks=== [[Media:Wisconsin-Madison2010_Notebook1.pdf]] [[Media:Wisconsin-Madison2010_Notebook2.pdf]] [[Media:Wisconsin-Madison2010_Notebook3.pdf]]
   Retrieved from "http://2010.igem.org/Team:Wisconsin-Madison/experiments"