Difference between revisions of "Part:BBa K4361106"
 
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<partinfo>BBa_K4361106 short</partinfo>
 
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This composite part shows our expression system for <i>E. coli</i> codon optimized BlcR with purification tag and cleavage site ([[Part:BBa_K4361103]]) in the pET-11a expression plasmid ([[Part:BBa_K4361105]]), for use in PURE reactions. We chose this system as it allowed us to express BlcR and test its properties in a single system (see also [[Part:BBa_K4361115]] and [[Part:BBa_K4361117]]).
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This composite part shows an expression system for <i>E. coli</i> codon-optimized genes with His-Tag purification tag ([[Part:BBa_K4361102]]) and TEV cleavage site ([[Part:BBa_K4361103]]). The backbone is the pET-11a expression plasmid ([[Part:BBa_K4361105]]). A pET11a expression system in E. coli is popular because it combines a high protein yield with good regulation over the expression. The T7 RNA polymerase has a lacUV5 promoter that is Isopropyl 𝛃-D-1-thiogalactopyranoside (IPTG) inducible. With the addition of IPTG, the gene upstream of the T7 promoter can be transcribed. This part does not represent the full sequence of pET-11a, but rather the plasmid after digestion with BamHI as it allows for the insertion of a new DNA sequence at the cleavage site.
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With this plasmid proteins can be effectively expressed in <i> E. coli</i>. The His-Tag allows His-Tag purification of the expressed protein. In addition, the TEV site can be used to cut off the His-Tag to exclude the interference of the tag with the activity of the protein.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:00, 12 October 2022


pET-11a with BlcR with 6xHis-tag and TEV protease cleavage site

This composite part shows an expression system for E. coli codon-optimized genes with His-Tag purification tag (Part:BBa_K4361102) and TEV cleavage site (Part:BBa_K4361103). The backbone is the pET-11a expression plasmid (Part:BBa_K4361105). A pET11a expression system in E. coli is popular because it combines a high protein yield with good regulation over the expression. The T7 RNA polymerase has a lacUV5 promoter that is Isopropyl 𝛃-D-1-thiogalactopyranoside (IPTG) inducible. With the addition of IPTG, the gene upstream of the T7 promoter can be transcribed. This part does not represent the full sequence of pET-11a, but rather the plasmid after digestion with BamHI as it allows for the insertion of a new DNA sequence at the cleavage site.

With this plasmid proteins can be effectively expressed in E. coli. The His-Tag allows His-Tag purification of the expressed protein. In addition, the TEV site can be used to cut off the His-Tag to exclude the interference of the tag with the activity of the protein.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 316
    Illegal XbaI site found at 5594
    Illegal PstI site found at 1068
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 316
    Illegal NheI site found at 5639
    Illegal NheI site found at 6446
    Illegal PstI site found at 1068
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 316
    Illegal BglII site found at 5528
    Illegal BamHI site found at 5672
    Illegal BamHI site found at 6581
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 316
    Illegal XbaI site found at 5594
    Illegal PstI site found at 1068
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 316
    Illegal XbaI site found at 5594
    Illegal PstI site found at 1068
    Illegal NgoMIV site found at 3394
    Illegal NgoMIV site found at 3748
    Illegal NgoMIV site found at 3908
    Illegal NgoMIV site found at 5496
    Illegal NgoMIV site found at 5830
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1242
    Illegal SapI.rc site found at 2324
    Illegal SapI.rc site found at 6341