References and Papers: Difference between revisions
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<li>[[BerkiGEMwet2009 | Home]]</li> | <li>[[BerkiGEMwet2009 | Home]]</li> | ||
<li>[[UCBigem09-Protocols | Protocols]]</li> | <li>[[UCBigem09-Protocols | Protocols]]</li> | ||
<li>[[Data| Data]]</li> | |||
<li>[[Notebooks | Notebooks]]</li> | <li>[[Notebooks | Notebooks]]</li> | ||
<li>[[Construction Files | ConstructionFiles ]] </li> | <li>[[Construction Files | ConstructionFiles ]] </li> | ||
<li id="current">[[References_and_Papers | References and Papers]]</li> | <li id="current">[[References_and_Papers | References and Papers]]</li> | ||
<li>[[Parts Database|Parts Database]] | |||
<li>[[UCBigem09-groupmtg|Group Mtg Presentations]] | |||
<li>[[About_Us | About Us]]</li> | <li>[[About_Us | About Us]]</li> | ||
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Here is an example of a cellulase being displayed by an Ice-Nucleation Protein displayer: | Here is an example of a cellulase being displayed by an Ice-Nucleation Protein displayer: | ||
doi:10.1016/S0141-0229(97)00224-X | doi:10.1016/S0141-0229(97)00224-X | ||
of particular note is the congo-red plate-based assay) | |||
DNS based assay for soluble and insoluble cellulose: http://www3.interscience.wiley.com/journal/107623737/abstract<br> | |||
Plate Based Screen: http://www3.interscience.wiley.com/journal/120054853/abstract | |||
Cellulose Binding Module (CBM) review: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1133952&blobtype=pdf | |||
---- | ---- | ||
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** XO loses FAD and can't be inhibited | ** XO loses FAD and can't be inhibited | ||
** Other inhibitors/competitors in liquid culture | ** Other inhibitors/competitors in liquid culture | ||
<br> | <br> | ||
NBT: | NBT: | ||
*Measure Absorbance at 560nm as a function of time in a sample containg SOD and a control sample | *Measure Absorbance at 560nm as a function of time in a sample containg SOD and a control sample | ||
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** 10 uL Riboflavin 4.4mg/100mL | ** 10 uL Riboflavin 4.4mg/100mL | ||
** they used 20 uL of purified SOD. | ** they used 20 uL of purified SOD. | ||
*The mixture is illuminated for 7 minutes and <math> A_560 </math> is measured. | * The mixture is illuminated for 7 minutes and <math> A_560 </math> is measured. | ||
* | |||
** | Something More Qualitative: | ||
* Could see if cells don't die in an environment containing reactive oxygen species. It would be easier then buying all of the reactants needed for qualitative measurements. | |||
* References: | |||
PMID: 3034103 | |||
---- | |||
'''EspA scfv''' | |||
If we are able to work with OH:157: | |||
* outer cell membrane fractions could be prepared from liquid cultures and a western blot could be preformed using the outer membrane expressed scfv. | |||
* Some sort of out growth assay based upon the ability of our cells to bind to immobilized OH:157 cells | |||
If we are not able to work with OH:157: | |||
* we will need to express a histidine (or streptavidin binding peptide) tagged espA in e. coli. This protein would need to be purified and imobilized on a column. | |||
* An ELISA could be preformed | |||
*References: | |||
PMID: 15243046 | |||
---- | |||
Latest revision as of 16:35, 14 August 2009
University of California Berkeley iGem 2009 Wetlab Team |
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Links
Post brief discussions of and links to interesting papers and references.
Functional Assays
Cellulase We're most interested in endo-1,4-beta-glucanases, as they are the rate limiting step in cellulose degradation. However, it may be necessary to include exocellulases to relieve product inhibition (this may or may not be true, see: doi:10.1016/0076-6879(88)60109-1 <--- suggests there is no synergism between exo's and endo's....
This is the sequencing paper from which we have taken all of our (Cellvibrio japonicus) "cellulases" up to this point: doi:10.1128/JB.01701-07
This is a Carboxymethylcellulose (CMC) assay (CMC is soluble) that is colorimetric: http://secure.megazyme.com/downloads/en/data/S-ACMC.pdf
Here is an example of a cellulase being displayed by an Ice-Nucleation Protein displayer: doi:10.1016/S0141-0229(97)00224-X of particular note is the congo-red plate-based assay)
DNS based assay for soluble and insoluble cellulose: http://www3.interscience.wiley.com/journal/107623737/abstract
Plate Based Screen: http://www3.interscience.wiley.com/journal/120054853/abstract
Cellulose Binding Module (CBM) review: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1133952&blobtype=pdf
Superoxide dismutase
Superoxide dismutase catalyzes the reaction:
[math]\displaystyle{ 2(O_2)^-+2H^+ --\gt (H_2)(O_2)+O_2 }[/math]
As the oxygen radical is too reactive to measure directly, all assays rely on the ability of SOD to compete with a free radical scavenger and inhibit a reaction with a chlorophore as a product. Here are a list of commonly used assays which would be appropriate in liquid cultures:
Cytochrome C:
- Measure inhibition of the initial rate of cytochrome C reduction under:
- 50 uM kPi
- 0.1 mM EDTA
- 50 uM Xanthine
- 10 uM ferricytochrome C
- Enough Xanthine Oxidase (~6nM) to cause an initial rate of absorption at 550nm of 0.025/min at pH 7.8 and 25 °C in a 3mL reaction can
- Problems:
- Need to establish the actual concentration of ferricytochrome C in stock solution.
- Cytochrome C contamination with SOD
- contamination of XO with lactoperoxidase
- XO loses FAD and can't be inhibited
- Other inhibitors/competitors in liquid culture
NBT:
- Measure Absorbance at 560nm as a function of time in a sample containg SOD and a control sample
- The following mixture is prepared:
- 27mL Buger (pH 7.8)
- 1.5 mL I-Methionine (300 mg/10)
- 1 mL NBT . 2HCL (14.1 mg/10mL)
- 750 uL Triton X-100 1% (w/v)
- 10 uL Riboflavin 4.4mg/100mL
- they used 20 uL of purified SOD.
- The mixture is illuminated for 7 minutes and [math]\displaystyle{ A_560 }[/math] is measured.
Something More Qualitative:
- Could see if cells don't die in an environment containing reactive oxygen species. It would be easier then buying all of the reactants needed for qualitative measurements.
- References:
PMID: 3034103
EspA scfv If we are able to work with OH:157:
- outer cell membrane fractions could be prepared from liquid cultures and a western blot could be preformed using the outer membrane expressed scfv.
- Some sort of out growth assay based upon the ability of our cells to bind to immobilized OH:157 cells
If we are not able to work with OH:157:
- we will need to express a histidine (or streptavidin binding peptide) tagged espA in e. coli. This protein would need to be purified and imobilized on a column.
- An ELISA could be preformed
- References:
PMID: 15243046
--- Tir This paper has some information about the binding between intimin and Tir: doi:10.1074/jbc.M401616200