Difference between revisions of "Part:BBa K3722009"
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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.
 
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.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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Biology
 
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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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Usage
+
===Biology===
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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.
+
SttH is a Tryptophan halogenases from Streptomyces toxytricini. It can substitute the 6-CH of Trp by a Bromine atom.
 +
<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/8/84/T--NWU-CHINA-A--part091.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
  
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Characterization
+
 
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  1. Identification
+
===Usage===
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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.
+
  Here we use BBa_K3722009 to verify the expression of SttH. We transformed the composite part BBa_K3722009 into E.coli DH5α&E.coli BL21(DE3)  to produce 6-Br-Trp. The positive clones were cultured.
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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  
+
===Characterization===
 +
1.Identification
 +
We received the synthesized DNA, then performed PCR with the primer of SttH. We used agarose gel electrophoresis to confirm that we got the right DNA fragment.  The experimental results were shown below and ideally fit the length we want.
 +
<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/b/ba/T--NWU-CHINA-A--part092.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 
 +
 
 +
 
 +
2.The proof of expression
 +
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  
 
E. coli BL21(DE3) bacterial culture which transformed only with pET-28a(+) as a control group,
 
E. coli BL21(DE3) bacterial culture which transformed only with pET-28a(+) as a control group,
−
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.
+
the expressed proteins were collected by centrifugation. Our target bands were observed through SDS-PAGE. The experimental results were shown below. We can easily find the target band which only shown in the left lane.
−
插入蛋白表达的图片
+
<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/1/19/T--NWU-CHINA-A--part093.png" width="100%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 
 +
 
 +
 
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 23:31, 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.


Biology

SttH is a Tryptophan halogenases from Streptomyces toxytricini. It can substitute the 6-CH of Trp by a Bromine atom.


Usage

Here we use BBa_K3722009 to verify the expression of SttH. We transformed the composite part BBa_K3722009 into E.coli DH5α&E.coli BL21(DE3)  to produce 6-Br-Trp. The positive clones were cultured.


Characterization

1.Identification We received the synthesized DNA, then performed PCR with the primer of SttH. We used agarose gel electrophoresis to confirm that we got the right DNA fragment. The experimental results were shown below and ideally fit the length we want.


2.The proof of expression 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 E. coli BL21(DE3) bacterial culture which transformed only with pET-28a(+) as a control group, the expressed proteins were collected by centrifugation. Our target bands were observed through SDS-PAGE. The experimental results were shown below. We can easily find the target band which only shown in the left lane.


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]