Difference between revisions of "Part:BBa K339010"

Revision as of 04:57, 30 October 2010 (view source) H.dastidar (Talk | contribs) (→‎Usage and Biology) ← Older edit		Latest revision as of 06:36, 30 October 2010 (view source) Emily Hicks (Talk | contribs)
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−	<h2>Derivation of the promoter</h2>
−	This promoter is found naturally in <i>E. coli</i>. This promoter is comprised of two naturally occuring promoters.
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−	<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Kraftpaperimage.png" border="0" alt="Kraft et al idea"></img>
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−	This image shows the plasmid schematic from paper by Kraft et al.
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−	In order to derive the promoter that is submitted, ibpAB (native) was blasted in order to locate the location in the  sequence provided in the paper.
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−	<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-clustal.png" border="0" alt="IbpAB"></img>
−	Clustal W alignment with native promoter region in front of ibpAB operon(P2_ibpAB)and the sequence provided in the paper (P1_proposed)
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−	Similarly, the FxsA promoter (native) was also aligned with the entire sequence in the paper.
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−	<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/FxsA.png" border="0" alt="FxsA"></img>
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−	These two alignments allowed us to derive the important DNA parts. There were parts eliminated. For example an internal PvoII site was eliminated as well at a T7 ribosome binding site was removed to use standard ribosome binding site. (B0034)
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−	<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Protein%20man/finalsequence.png" border="0" alt="ibpAB"></img>
	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>

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Latest revision as of 06:36, 30 October 2010

ibpAB

Inclusion Body Binding Protein/ FxsA fusion Promoter

Usage and Biology

This is a fusion promoter which fuses two heat shock promoters from E. Coli: Fxsa and ibpAB. Kraft et al (1997) designed this fusion promoter and found it to be considerably more sensitive to misfoled protein in the cytoplasm than either of the promoters alone.

The Fxsa promoter is not well documented. The ibpAB promoter controls the transcription of two small proteins: ibpA and ibpB. These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures. The binding of these proteins to the misfolded protein lowers its hydrophobicity, preventing further binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005). Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993).

http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-2.png"

B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)

Sequence and Features

Functional Parameters

Characterization of the ibpAB-fsxA fusion promoter's response to properly folding and misfolding proteins

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"

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.