Team:Wisconsin-Madison/experiments
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- | + | <li> Measure OD600 | |
- | + | <li> To inactivate EPS-degrading enzymes and completely release EPS from cell surface: | |
- | + | <ul> | |
- | + | <li> Boil sample for 15 min | |
- | + | <li> Cool to room temp | |
- | # Add three volumes of ethanol to 40 ml of supernatant fraction | + | <li> Centrifuge at 14,000g for 30 min at 4°C |
- | # Place in 4°C overnight | + | </ul> |
- | # Centrifuge at 14,000g for 30 min at 4°C | + | <li> # Add three volumes of ethanol to 40 ml of supernatant fraction |
- | # Dissolve pellet in 1 ml of sterile distilled water | + | <li> # Place in 4°C overnight |
+ | <li> # Centrifuge at 14,000g for 30 min at 4°C | ||
+ | <li># Dissolve pellet in 1 ml of sterile distilled water | ||
# Quantifications: Use negative controls of glucose and 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 | ## Add 100 ul of colonic acid preparation to 1 ml of sterile distilled water |
Revision as of 23:48, 29 September 2010
Enzyme Treatment
Encapsulation
Quantification of Colonic Acid Production
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 |
- Measure OD600
- 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
- # Add three volumes of ethanol to 40 ml of supernatant fraction
- # Place in 4°C overnight
- # Centrifuge at 14,000g for 30 min at 4°C
- # 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]]