Team:Wisconsin-Madison/experiments
From 2010.igem.org
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===Encapsulation=== | ===Encapsulation=== | ||
====Quantification of Colonic Acid Production==== | ====Quantification of Colonic Acid Production==== | ||
- | + | {| 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''' | ||
+ | |- | ||
+ | |<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 | # Measure OD600 | ||
# To inactivate EPS-degrading enzymes and completely release EPS from cell surface: | # To inactivate EPS-degrading enzymes and completely release EPS from cell surface: | ||
Line 45: | Line 67: | ||
====Cell Survivability Testing==== | ====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''' | ||
+ | |- | ||
+ | |<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==== | ====Best Combination==== | ||
<br> | <br> |
Revision as of 23:41, 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
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