Difference between revisions of "Part:BBa K3722009"
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===Usage and Biology===
 
===Usage and Biology===
 
Biology
 
Biology
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TnaA cleaves 6-Br-Trp into 6-Br-indole , pyruvate and ammonia. So we can use this composite part to synthetic 6-Br-indole as the second proceed to product 6,6’-dibromo indigo.
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SttH is a Tryptophan halogenases from Streptomyces toxytricini . It can substitute the 6-CH of Trp into a Bromine atom.
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  有图片
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Usage
 
Usage
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We construct the composite part BBa_K3332052 and transformed it into E. coli BL21 (DE3) to verify its expression.
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Here, we use BBa_K3722009 to verify the expression of SttH. We transformed the composite part BBa_K3722009 into E. coli BL21(DE3) to produce 6-Br-Trp. The positive clones were cultivated.
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Characterization
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1. Identification
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WE received the synthesized DNA, then did PCR with the primer of SttH. We use agarose gel electrophoresis to confirm we get the right DNA fragment.  the experimental results were shown below and ideally fit the length we want.
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插入PCR图片
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2. The proof of expression
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  We connected the composite part BBa_K3722009 with pET-28a(+) , then transformed it into E. coli BL21(DE3). we added 0.1 mM IPTG and incubated the bacterial culture at 18℃ for 16 hours to get a high expression with low noise protein. We also incubated the other
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E. coli BL21(DE3) bacterial culture which transformed only with pET-28a(+) as a control group,
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the expressed proteins were collected by centrifugation. Our target bands are observed through SDS-PAGE. the experimental results were shown below. We can easily find the target band which only shown in the left line.
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插入蛋白表达的图片
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3722009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3722009 SequenceAndFeatures</partinfo>
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Revision as of 08:57, 21 October 2021


T7_Promoter-RBS-SttH-T7_Terminator

Flavin-dependent Trp halogenases use halide ions (X−) and FADH2 for halogenation，if you use it in cell-free systems, requiring supplementation of the high-cost cofactor FADH2 and additional cofactor regenerating enzymes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1635
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 268
    Illegal NgoMIV site found at 526
    Illegal AgeI site found at 755
  • 1000
    COMPATIBLE WITH RFC[1000]