Difference between revisions of "Part:BBa K638202"
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Arabinose inducible Poly-His Reflectin A1 generator produces a poly-histidine (his-tagged) form of Reflectin A1([[Part:BBa_K638001 | BBa_K638001]]).  
 
Arabinose inducible Poly-His Reflectin A1 generator produces a poly-histidine (his-tagged) form of Reflectin A1([[Part:BBa_K638001 | BBa_K638001]]).  
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Bacteria expressing this construct will produce Reflectin A1 with a poly-histidine tag at the N-terminus in order to enable purification with a his-affinity column used in conjunction with nickel or cobalt resins.  
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Bacteria expressing this construct will produce Reflectin A1 with a poly-histidine tag at the N-terminus (upon induction with arabinose) in order to enable purification with a his-affinity column used in conjunction with nickel or cobalt resins.  
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The Reflectin A1 gene is under the control of the pBAD promoter [[Part:BBa_I0500 | BBa_I0500]], which is tightly controlled by two factors:
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*'''L-arabinose''' monosaccharide taken up by the cell from the medium, which acts as an ''inducer''.
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*'''AraC''' protein included in the I0500 biobrick, which acts an a ''repressor''.
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Therefore, the araC-pBAD system offers regulatable control of gene expression in the presence of the inducer and highly repressed in the absence of the inducer.
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Read more about [http://2011.igem.org/Team:Cambridge/Experiments/Low_Level_Expression the pBAD and arabinose system in E.coli].
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Revision as of 23:35, 21 September 2011

Arabinose inducible Poly-His Reflectin A1 generator

Arabinose inducible Poly-His Reflectin A1 generator produces a poly-histidine (his-tagged) form of Reflectin A1( BBa_K638001).

Bacteria expressing this construct will produce Reflectin A1 with a poly-histidine tag at the N-terminus (upon induction with arabinose) in order to enable purification with a his-affinity column used in conjunction with nickel or cobalt resins.

The Reflectin A1 gene is under the control of the pBAD promoter BBa_I0500, which is tightly controlled by two factors:

  • L-arabinose monosaccharide taken up by the cell from the medium, which acts as an inducer.
  • AraC protein included in the I0500 biobrick, which acts an a repressor.

Therefore, the araC-pBAD system offers regulatable control of gene expression in the presence of the inducer and highly repressed in the absence of the inducer. Read more about [http://2011.igem.org/Team:Cambridge/Experiments/Low_Level_Expression the pBAD and arabinose system in E.coli].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 1261
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961

Usage and Biology

This device was characterised as Part:BBa_K638301, where reflectin is fused to GFP as an easy reporter for microscopy. We assessed folding on multiple backbones.

  • Reflectin-GFP on pSB1A3(a high copy plasmid), induced with 1mM Arabinose. Fluorescence is localised in dots at the end of the cells, indicating that fusion protein is localised to insoluble inclusion bodies.

  • Reflectin-GFP on pSB3K3 (low/med copy plasmid), induced with 1mM Arabinose. The fusion protein is distributed throughout the cytoplasm, indicating soluble protein is expressed.

From this confocal imaging, we recommend that reflectin be expressed on a low/med copy number backbone to allow protein folding to occur to give soluble protein. High copy number plasmids give inclusion bodies. Some inclusion bodies are also visible in the low copy construct, suggesting that some cells are expressing more fusion protein than others. See our experience of the pBAD promoter for a discussion of variations of induction within a population of cells.

Safety

The protein coding sequence for reflectin originally came from cells of an edible squid. There have been no reported safety issues for reflectins, so we do not anticipate the need for extra precautions when using this BioBrick part. See our [http://2011.igem.org/Team:Cambridge/Safety safety page] for more information.