Part:BBa_K339003
malE31ΔSS
Mutant form of the Maltose binding protein (malE) with signal sequence deleted.
Usage and Biology
This mutant form of the maltose binding protein has the signal sequence deleted (amino acids). This results in the protein not benig localized to the periplasm of the cell, but rather staying in the cytoplasm of the cell. This mutant also has a BamHI site located at codon 32-33 replacing the wild-type sequence Gly-Ile. This two amino acid mutation destroys the folding capabilities of the protein, resulting in a non-folder.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 360
Illegal BamHI site found at 96 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 58
Functional Parameters
Characterization of the ibpAB-fsxA fusion promoter's response to properly folding and misfolding proteins</h2></P>
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"
<p> This graph shows the GFP fluorescence produced when MalEΔSS and MalE31ΔSS downstream of maltose-inducible promoters were transformed into Top10 competent cells containing the ibpAB-fsxA promoter coupled with a GFP reporter.
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.
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