References and Papers
University of California Berkeley iGem 2009 Wetlab Team
Post brief discussions of and links to interesting papers and references.
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 catalyzes the reaction:
2(O2) − + 2H + − − > (H2)(O2) + O2
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:
- 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
- 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
- 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 A560 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.
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
--- Tir This paper has some information about the binding between intimin and Tir: doi:10.1074/jbc.M401616200