Revision as of 09:35, 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. tetsing with known folding and misfolding proteins
  2. tetsing with NLPE, an outer membrane lipoprotein known to activate the cpx pathway
  3. tetsing with varying temperature conditions

Testing with known folding and misfolding proteins

We first needed to identify proteins that we know fold and don’t fold well inE. coili. For this we chose the maltose binding protein. 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 and is classified as a non-folder. MalE with the signal sequence removed, does not move in to the periplas, 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 coverning folding and non-folding in botht he periplas and the cytoplasm.

maltose binding chart place holder

We received these genes from the Betton labs in France. We biobricked these parts, but before testing our stress reporters with them, we wanted to first test these parts to sohow that they work as expected.. 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