Revision as of 15:54, 27 October 2010 by H.dastidar (Talk | contribs)

Testing our system

Once constructed, we needed a way to test the cytoplasm and periplasmic stress promoters in order to characterize them. We did this in three different ways.

  1. testing with known folding and misfolding proteins
  2. testing with NlpE, an outer membrane lipoprotein known to activate the Cpx regulon
  3. testing with varying temperature conditions

Testing with known folding and misfolding proteins

First it was necessary to identify proteins that are known folders and known non-folders in E. coil. Maltose binding protein was selected for this purpose. Wild type maltose binding protein (MalE) is transporter protein that is shuttled to the periplasmic space of E.coli. This is a protein known to fold extremely well in the periplasm of E. Coli. MalE31, a mutant with two amino acid substitutions at postion 33 and 34, does not fold in the periplasmic and is classified as a non-folder. MalE,requires a signal sequence in order to be transported to the periplasmic space, with signal sequence removed, does not move in to the periplasm, but remains in the cytoplasm where it folds extremely well. MalE31 with the signal sequence removed, is a non folder in the cytoplasm. Thus we have four proteins covering folding and non-folding in both the periplasm and the cytoplasm.

maltose binding chart place holder

The wild type MalE as well as the mutant versions were received from the Betton lab in France. These parts were biobricked, but prior to testing the selected stress reporter circuits with these parts, it was necessary to test these MalE variations to ensure that they were functional and matched literature data. To do this, we transformed them into strains of cells containing cpxR and degP promoters up stream of a lacZ rpeorter (Raivio labs). We would expect malE31, if it misfolded, to activate the cpxR and degP stress promoters, thsus providing a blue output from lacZ. MalE on the other hand would not misfod, and therefore would not activate these promoters, and we would not expect to see any lacZ activity. This allowed us to conlcude that malE and malE31 work the way that we expected them to. See results on our characterization page.

Once malE and malE31 were shown to be functional, we then used them to test out the stress promoters. We did this by making competent cells containing our reporeter circuits. We then transofmed in exprressio constructs for our malE and mutant malE proteins. We then measured fluorescence output from our reporter constructs. See resuts for this on our characterization opage.

Testing with NLPE

NLPE is an outer membrane lipoprotein that literature has shown actibates the cpX pathway. We transformed expression costructs for this protien (obtained from the Rvaio lab) into competent cells containing our cpxR reporter and looked for fluoresecnt output. Results for this experiment can be viewed on our characyerization page.

Testing with Varying Temperature Conditions

Finally we tested the cpxR promoter