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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
| 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
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
