Part:BBa_K1188002
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4
Illegal AgeI site found at 223 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters: Austin_UTexas
Burden Imposed by this Part:
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.
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