Difference between revisions of "Part:BBa K1188002"
 
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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.
 
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.
  
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Experimentally results show that calcium concentrations of 10 mM or above results in effective precipitation while conserving the function of the purified protein, in the the case of GFP.  Calcium concentrations below ~1 mM do not produce a useful amount of precipitate.
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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.
  
 
[[File:RTXPrecipitation.PNG|200px]]
 
[[File:RTXPrecipitation.PNG|200px]]
 
[[File:RTXSDSPage.PNG|200px]]
 
[[File:RTXSDSPage.PNG|200px]]
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SDS-PAGE - From Left to Right:
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Lane 1: Clarified cell lysate
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Lane 2: 0mM supernatant after addition of 0mM CaCl2 and 2min spin at 13.2k RPM
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Lane 3: 0mM supernatant after wash and resuspension in 50mM trisHCl with 0mM EDTA
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Lane 4: 0.1mM supernatant after addition of 0.1mM CaCl2 and 2min spin at 13.2k RPM
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Lane 5: 0.1mM supernatant after wash and resuspension in 50mM trisHCl with 0.1mM EDTA
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Lane 6: 1mM supernatant after addition of 1mM CaCl2 and 2min spin at 13.2k RPM
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Lane 7: 1mM supernatant after wash and resuspension in 50mM trisHCl with 1mM EDTA
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Lane 8: 10mM supernatant after addition of 10mM CaCl2 and 2min spin at 13.2k RPM
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Lane 9: 10mM supernatant after wash and resuspension in 50mM trisHCl with 10mM EDTA
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Lane 10: 100mM supernatant after addition of 100mM CaCl2 and 2min spin at 13.2k RPM
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Lane 11: 100mM supernatant after wash and resuspension in 50mM trisHCl with 100mM EDTA
  
 
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===Functional Parameters===
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==Functional Parameters: Austin_UTexas==
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<html>
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<body>
 
<partinfo>BBa_K1188002 parameters</partinfo>
 
<partinfo>BBa_K1188002 parameters</partinfo>
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<!-- -->
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<h3><center>Burden Imposed by this Part:</center></h3>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
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</div>
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<figcaption><center><b>Burden Value: 1.0 ± 1.4% </b></center></figcaption>
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</figure>
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<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the
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<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods, an explanation of the sources of burden,  and other conclusions from a large-scale measurement project conducted by the <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p>
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<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
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</body>
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</html>

Latest revision as of 19:01, 27 August 2020

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]



Functional Parameters: Austin_UTexas

BBa_K1188002 parameters

Burden Imposed by this Part:

Burden Value: 1.0 ± 1.4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.