Difference between revisions of "Part:BBa K1162009"

 
 
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<partinfo>BBa_K1162009 short</partinfo>
 
<partinfo>BBa_K1162009 short</partinfo>
  
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This part uses assembly standard 23 (RFC 23-silver fusion) to allow for protein fusions. This 10x His Tag is similar to BBa_K844000 but has the double stop codon (TAA TAA) removed unlike BBa_K844000. (https://parts.igem.org/wiki/index.php?title=Part:BBa_K844000).  
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===Design===
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Another characteristic of this part is the presence of a Met (ATG) for direct His Tag and protein expression. Since the presence of the Met (ATG) this His Tag should be used for protein purification at the N-terminal. The user can place a promoter-rbs system of their choice in front of this His Tag for direct expression. The lack of any stop codon in this His Tag allows the user to add additional features downstream of the gene e.g. secretion tag or GFP (note, the gene itself must not contain any stop codons if the His Tag is to be used like this).
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Related parts
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This part was designed with [https://parts.igem.org/Assembly_standard_23 Assembly Standard #23 (Silver Fusion)] to allow for protein fusions. This 10x His-Tag is an improvement upon [https://parts.igem.org/wiki/index.php?title=Part:BBa_K844000 BBa_K844000] by removing the double stop codon (TAA TAA) to allow for C-terminal fusion of proteins. Another key characteristic of this part is the presence of a Methionine(atg) at the start of the coding region (N-terminal), which enables the user to fuse proteins downstream and then purify. The promoter-rbs system of choice can be placed in front of this His-Tag for protein expression.
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BBa_K844000 (https://parts.igem.org/wiki/index.php?title=Part:BBa_K844000), this link also provides an extensive review of existing Histidine Tags in the Parts Registry.
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===Experience===
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https://static.igem.org/mediawiki/2013/1/10/AtgHTGFP.png
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<br>
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<b>Figure 1.</b> The above image shows a GFP dot image (on parafilm, illuminated by UV light) in which it is clear that the N-terminal 10x His-Tag (BBa_K1162009) method of purification works as desired and adds another important purification system to the registry.
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<br>
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https://static.igem.org/mediawiki/2013/1/1c/AtgHTGFP_protein_gel.PNG
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<br>
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<b>Figure 2.</b> From the SDS-PAGE gel it can be seen that there is pure GFP (~26.9 kDa) in elution fractions number 2 and 3. The wash fractions do not appear to have any GFP, which indicates that the N-terminal 10x His-Tag is strongly bound to the Nickel resin column during washing steps.  
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===Related parts===
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[https://parts.igem.org/wiki/index.php?title=Part:BBa_K844000 BBa_K844000] - this link also provides an extensive review of existing Histidine Tags in the Parts Registry.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:51, 28 September 2013

10x-Histidine (10x-His) Tag with Met (ATG) For N-terminal purification

Design

This part was designed with Assembly Standard #23 (Silver Fusion) to allow for protein fusions. This 10x His-Tag is an improvement upon BBa_K844000 by removing the double stop codon (TAA TAA) to allow for C-terminal fusion of proteins. Another key characteristic of this part is the presence of a Methionine(atg) at the start of the coding region (N-terminal), which enables the user to fuse proteins downstream and then purify. The promoter-rbs system of choice can be placed in front of this His-Tag for protein expression.

Experience

AtgHTGFP.png
Figure 1. The above image shows a GFP dot image (on parafilm, illuminated by UV light) in which it is clear that the N-terminal 10x His-Tag (BBa_K1162009) method of purification works as desired and adds another important purification system to the registry.
AtgHTGFP_protein_gel.PNG
Figure 2. From the SDS-PAGE gel it can be seen that there is pure GFP (~26.9 kDa) in elution fractions number 2 and 3. The wash fractions do not appear to have any GFP, which indicates that the N-terminal 10x His-Tag is strongly bound to the Nickel resin column during washing steps.

Related parts

BBa_K844000 - this link also provides an extensive review of existing Histidine Tags in the Parts Registry.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]