References and Papers

University of California Berkeley iGem 2009 Wetlab Team

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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)

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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

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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

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--- Tir This paper has some information about the binding between intimin and Tir: doi:10.1074/jbc.M401616200