Difference between revisions of "Part:BBa K339002"

Latest revision as of 23:40, 30 October 2010

malEΔSS

This is a form of the maltose binding protien (malE) native to E. Coli, that has the signal sequence removed.

Usage and Biology

With the signal sequence deleted, this protein is not localized to the periplasm, but rather stays in the cytoplasm. Despite this, it is still able to fold well in the cytoplasm (Betton et al., 2002). This protein can therefore be used as a fusion partner for hard to express proteins that stay in the cytoplasm of the cell. This part can also be used to characterize cytoplasmic misfolding promoters.

Sequence and Features

Functional Parameters

Characterization of malE folding ability in the cytoplasm

Purpose/ Protocol

The purpose of this assay is to characterize the ibpAB fusion promoter (ibpAB-FxSA) with a protein that is known to fold correctly (MalEΔSS) and with a protein that are known to misfold (MalE31ΔSS) while remaining in the cytoplasm. The promoter was coupled with green fluorescent protein so when activated, the reporter would be produced. A construct received from Jean-Michel Betton's lab containing MalEΔSS and MalE31ΔSS downstream from a maltose-induced promoter were transformed into Top10 competent cells containing the plasmid with the ibpAB-fsxA GFP reporter plasmid (ibpAB-I13504). Overnight cultures were made from these transformations in 5 mL of LB Lennox Broth and left to grow for sixteen hours. Induction was done with multiple concentrations of maltose to produce different quantities of protein and the cells were shaken at 30°C. Four hours after induction, GFP fluorescence was measured and can be seen below.

Results

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-data.png"

Discussion and Conclusions

The graph trend lines show a sharp decrease from the opening GFP output before rising again to level off. The induction using varying concentrations of maltose to produce known misfolding protein shows that the properly folding maltose binding protein (MalEΔSS) causes more fluorescence output than the misfolding maltose binding protein (MalE31ΔSS). This is in contradiction with the literature data. However, the last data value at maltose concentration of 0.5% maltose added to the solution is what creates this. In the future, more characterization assays should be run with inductions using more variants of maltose concentrations. These would allow for further verification and confirmation of either literature data or our data. There have been very few studies using this fusion promoter so little is known about it. In addition, more known folding and misfolding proteins can be used to enhance the data gained with maltose binding protein.