Tag

Part:BBa_K1188002

Designed by: Jean-Francois Lalonde   Group: iGEM13_CU-Boulder   (2013-08-23)
Revision as of 15:35, 27 September 2013 by Pk197 (Talk | contribs)

RTX - Calcium Binding Protein

RTX (repeats-in-toxin) is a polypeptide motif consisting of a repeating sequence of amino acids. It undergoes a conformation change in the presence of calcium which causes it to precipitate from solution. This is the shorter (8-mer) version of this part, and is in Freiburg format. This allows RTX to be fused to itself to create a longer RTX chain, or to other proteins which can then be precipitated from a solution. This is useful for the purpose of protein purification, or alternatively to remove an unwanted protein from a solution.

Experimental results show that calcium concentrations of 10 mM or above result in effective precipitation and is capable of a high level of purification of a fused protein (in this case, GFP) without disrupting protein function (green fluorescence is present). Additionally, calcium concentrations below ~1 mM do not produce a useful amount of precipitate for the purpose of protein purification.

RTXPrecipitation.PNG RTXSDSPage.PNG

SDS-PAGE - From Left to Right:

Lane 1: Clarified cell lysate

Lane 2: 0mM supernatant after addition of 0mM CaCl2 and 2min spin at 13.2k RPM

Lane 3: 0mM supernatant after wash and resuspension in 50mM trisHCl with 0mM EDTA

Lane 4: 0.1mM supernatant after addition of 0.1mM CaCl2 and 2min spin at 13.2k RPM

Lane 5: 0.1mM supernatant after wash and resuspension in 50mM trisHCl with 0.1mM EDTA

Lane 6: 1mM supernatant after addition of 1mM CaCl2 and 2min spin at 13.2k RPM

Lane 7: 1mM supernatant after wash and resuspension in 50mM trisHCl with 1mM EDTA

Lane 8: 10mM supernatant after addition of 10mM CaCl2 and 2min spin at 13.2k RPM

Lane 9: 10mM supernatant after wash and resuspension in 50mM trisHCl with 10mM EDTA

Lane 10: 100mM supernatant after addition of 100mM CaCl2 and 2min spin at 13.2k RPM

Lane 11: 100mM supernatant after wash and resuspension in 50mM trisHCl with 100mM EDTA

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
    Illegal AgeI site found at 223
  • 1000
    COMPATIBLE WITH RFC[1000]


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