Difference between revisions of "Part:BBa K3440007"

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===Usage and Biology===
 
===Usage and Biology===
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Expresses AiiA under constitutive promoter. AiiA degrades lactones.
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This part expresses AiiA under constitutive promoter. AiiA is a lactonase originally from Bacillus sp (UniProtKB - Q9L8R8) and it degrades lactones (AHLs) such as the ones we use in our project, C4-HSL and C6-HSL.
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In our project, AiiA is used as a negative feedback loop in our electricity output module in Shewanella to degrade AHLs used as quorum sensing molecules. Those AHLs are also in a positive feedback loop, thus creating oscillations.
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We used part BBa_K3440007 to prove that AiiA could be expressed under constitutive promoter BBa_J23100.
  
 
===Characterization===
 
===Characterization===
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Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.
 
Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.
  
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[[File:gelGJ.png|thumb|center|500px|Colony PCR gel for BBa_K3440007(G) and BBa_K3440008(J)]]
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After heat shock transformation of pSB1C3 plasmids containing BBa_K3440007 in E. coli TOP 10 cells, we picked colonies from plates (Figure 1), PCR amplified them and ran gels 30 min at 180V (Figure 2). We obtained two bands with a seemingly correct size (H9 and H13 for a size of 1189bp).
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[[File:BBa_K3440000_WB.png|thumb|center|500px|Western blot with G7 as BBa_K3440007]]
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<div><ul>
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<li style="display: inline-block;vertical-align: top;">[[File: T--Stockholm--PlateH.png|thumb|left|300px|Figure 1: Transformation plate of BBa_K3440007 (noted H)]]</li>
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<li style="display: inline-block;vertical-align: top;">[[File: T--Stockholm-Gel_BBa_K3440007.png|thumb|center|500px|Figure 2: colony PCR gel of BBa_K3440007 (H)]]</li>
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</ul></div>
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However, when we sent those samples for Sanger sequencing to Microsynth AG, it appeared that none of them had the correct sequence. We therefore highly recommend other teams to always send their samples for sequencing, even when the bands on gels have the right size.
  
 
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Latest revision as of 23:19, 27 October 2020


AiiA-Myc under constitutive promoter

Pconst(BBa_J23100)-RBS(BBa_B0034)-AiiA(BBa_C0060)-Myc(BBa_K823036)

Usage and Biology

This part expresses AiiA under constitutive promoter. AiiA is a lactonase originally from Bacillus sp (UniProtKB - Q9L8R8) and it degrades lactones (AHLs) such as the ones we use in our project, C4-HSL and C6-HSL. In our project, AiiA is used as a negative feedback loop in our electricity output module in Shewanella to degrade AHLs used as quorum sensing molecules. Those AHLs are also in a positive feedback loop, thus creating oscillations.

We used part BBa_K3440007 to prove that AiiA could be expressed under constitutive promoter BBa_J23100.

Characterization

Due to the pandemics, we haven’t been able to use biobricks to create the iGEM Stockholm 2020 parts. Those parts were ordered as gene blocks from Integrated DNA Technologies Inc.. As a result, the sequences of the biobricks used are the same, but the scars between biobricks might differ, as well as the final size of the part.

After heat shock transformation of pSB1C3 plasmids containing BBa_K3440007 in E. coli TOP 10 cells, we picked colonies from plates (Figure 1), PCR amplified them and ran gels 30 min at 180V (Figure 2). We obtained two bands with a seemingly correct size (H9 and H13 for a size of 1189bp).

  • Figure 1: Transformation plate of BBa_K3440007 (noted H)
  • Figure 2: colony PCR gel of BBa_K3440007 (H)

However, when we sent those samples for Sanger sequencing to Microsynth AG, it appeared that none of them had the correct sequence. We therefore highly recommend other teams to always send their samples for sequencing, even when the bands on gels have the right size.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 907
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 160
  • 1000
    COMPATIBLE WITH RFC[1000]