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Team:Wisconsin-Madison/experiments
From 2010.igem.org
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'''Background''' | '''Background''' | ||
Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content. | Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content. | ||
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<br> | <br> | ||
'''Materials''' | '''Materials''' | ||
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See entire procedure : [[Media:UW_colonicacidassay.pdf|Download]] | See entire procedure : [[Media:UW_colonicacidassay.pdf|Download]] | ||
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| + | See original reference: [[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link1]],[[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link2]][[http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link3]] | ||
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====Cell Survivability Testing==== | ====Cell Survivability Testing==== | ||
| - | {| border="1" cellpadding="5" cellspacing="0" align="center" | + | {| border="1" cellpadding="5" cellspacing="0" align="center" font-size="80%" |
|+ align="center" style="color:#000000;" |''Parts used in this experiment'' | |+ align="center" style="color:#000000;" |''Parts used in this experiment'' | ||
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|Inducible - IPTG | |Inducible - IPTG | ||
|502 | |502 | ||
| + | |- | ||
| + | |<partinfo>BBa_k200021</partinfo> | ||
| + | |Empty Vector/Contol | ||
| + | |Inducible - IPTG | ||
| + | |NA | ||
|} | |} | ||
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<br> | <br> | ||
===Inducible-Repressible Expression=== | ===Inducible-Repressible Expression=== | ||
Revision as of 03:24, 20 October 2010
Enzyme Treatment
Encapsulation
Colonic Acid Quantification
| 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 |
| <partinfo>BBa_k200021</partinfo> | Empty Vector/Contol | Inducible - IPTG | NA |
Background
Colonic Acid is a polysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate. Biological extracts often contain compounds, which under heating with H2SO4 yield brown products absorbing between 396 nm and 427 nm. Colonic acid can be estimated by measuring L-fucose content.
Materials
- H2SO4/H2O (6:1 v/v)
- Cysteine hydrochloride
- L-Frucose (calibration curve)
Cell Culture: (Final 50ml sample)
- Transform plasmids for high protein production
- Incubate plate overnight (do not place in fridge before next step)
- Prepare liquid culture of each sample
- Induce with 1mM IPTG at OD600 = 0.05
- Grow cells to stationary phase
Protocol:
- Prepare overnight liquid culture of 50 ml sample
- 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 70% 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
Quantification: Use negative controls of glucose and sterile distilled water
- Mix 4.5 ml of H2SO4/H2O (6:1 v/v) with 100uL of 1ml sample
- Heat mixture to 100°C for 20 min
- Cool to room temperature
- Measure absorbance at 396 nm and 427 nm
- Add 100 μL of 10M 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
L-Fucose – Standard Curve
- Concentrations of L-Fucose ranging from 0 μg/ml to 100 μg/ml
- L-Fucose was added to the appropriate amount of sterile distilled water to result in the following concentrations and 1ml of this preparation was taken from step 8(b) to 8(h) from Colonic Acid Quantification Assay (above)
See entire procedure : Download
See original reference: http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link1,http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link2http://pubs.acs.org/doi/abs/10.1021/ja01129a015 Link3
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 |
| <partinfo>BBa_k200021</partinfo> | Empty Vector/Contol | Inducible - IPTG | NA |
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
