Difference between revisions of "Part:BBa K3722011"

 
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Linking MaFMO and TnaA together can achieve the simultaneous expression of two enzymes in one cell, greatly improving the efficiency of the reaction.
 
Linking MaFMO and TnaA together can achieve the simultaneous expression of two enzymes in one cell, greatly improving the efficiency of the reaction.
  
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===Usage and Biology===
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===Biology===
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This part can achieve the highest yield of producing and simply the reaction process. Only one kind of engineering bacteria can  product 6,6’-dibromo indigo.
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<html>
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    <figure>
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        <img src="https://2021.igem.org/wiki/images/9/90/T--NWU-CHINA-A--part111.png" width="50%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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===Usage===
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We ligased TnaA (BBa_K3722000) and MaFMO (BBa_K3722002) on the expression vector pET-28a(+) to get this composite part by In-Fusion. Then we transformed it into E. coli DH5α & E. coli BL21(DE3).
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===Characterization===
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1.The verification of the expression
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<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/d/d3/T--NWU-CHINA-A--part112.png" width="50%" style="float:center">
 +
        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig.1''' SDS-PAGE analysis of MaFMO .Induction condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 18°C and 200 rpm.(1:negitive control  2: 3.5h  3:9h  4:10h  5:11h  6:12h)
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2.Productive result
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Excellent production effect was obtained after adding 6-Br-Trp.
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<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/8/8c/T--NWU-CHINA-A--part113.jpg" width="50%" style="float:center">
 +
        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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We compared two reaction systems by Microplate Reader, and found BBa_K3722011 was more productive. We suspected that this is due to the transmenmbrane transport of 6-Br-indole was skipped.
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 +
<html>
 +
    <figure>
 +
        <img src="https://2021.igem.org/wiki/images/b/b8/T--NWU-CHINA-A--part114.png" width="50%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
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    </figure>
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</html>
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:'''Fig.2''' The absorption curves of Tyrian Purple production.
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Revision as of 00:37, 22 October 2021


T7_Promoter-RBS-His Tag-MaFMO-RBS-His Tag-TnaA-T7_Terminator

Linking MaFMO and TnaA together can achieve the simultaneous expression of two enzymes in one cell, greatly improving the efficiency of the reaction.


Biology

This part can achieve the highest yield of producing and simply the reaction process. Only one kind of engineering bacteria can product 6,6’-dibromo indigo.

Usage

We ligased TnaA (BBa_K3722000) and MaFMO (BBa_K3722002) on the expression vector pET-28a(+) to get this composite part by In-Fusion. Then we transformed it into E. coli DH5α & E. coli BL21(DE3).


Characterization

1.The verification of the expression

Fig.1 SDS-PAGE analysis of MaFMO .Induction condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 18°C and 200 rpm.(1:negitive control 2: 3.5h 3:9h 4:10h 5:11h 6:12h)

2.Productive result Excellent production effect was obtained after adding 6-Br-Trp.

We compared two reaction systems by Microplate Reader, and found BBa_K3722011 was more productive. We suspected that this is due to the transmenmbrane transport of 6-Br-indole was skipped.

Fig.2 The absorption curves of Tyrian Purple production.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2940
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]