Difference between revisions of "Part:BBa K3440010"

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(→‎Usage and Biology)
 
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<partinfo>BBa_K3440010 short</partinfo>
 
<partinfo>BBa_K3440010 short</partinfo>
  
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Pbphr1(BBa_K1155001)-RBS(BBa_B0034)-LuxI(BBa_C0061)-Myc(BBa_K823036)
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Pbphr1(BBa_K1155001) - RBS(BBa_B0034) - LuxI(BBa_C0061) - Myc(BBa_K823036)
  
 
===Usage and Biology===
 
===Usage and Biology===
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Expresses LuxI under bphR1 promoter. bphR1 promoter can be activated by bphR2 or the complex bphR2:PCB. LuxI produces 3OC6-HSL, a quorum sensing molecule.
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This part expresses LuxI under bphR1 promoter.  
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bphR1 promoter is found in Pseudomonas pseudoalcaligenes KF707 and regulates the transcription of a series of biphenyl catabolic genes. In our project, we rely on this promoter for the detection of polychlorinated biphenyls, pollutants similar to dioxins and found in the Baltic Sea. In particular, in our final system, bphR1 promoter is activated by the complex bphR2:PCB, with bphR2 expressed constitutively and PCB coming from the analysed sea water.  
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LuxI is originally found in Vibrio fischeri (UniProtKB - P12747) and produces 3OC6-HSL, a quorum sensing molecule used in our system to communicate between our detection module in E. coli and our electrical output module in Shewanella oneidensis.
 +
In our project, the BBa_K3440010 part is used in order to prove that LuxI can be expressed under bphr1 promoter in the presence of bphR2 and PCBs and to test the leakiness of bphR1 (also tested with BBa_K3440001). A myc tag was added to facilitate characterization.
  
 
===Characterization===
 
===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.
 
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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We sent this part for sequencing to Microsynth AG. The sequence obtained corresponded to the expected part.  
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[[File:gelML.png|thumb|center|500px|Colony PCR gel for BBa_K3440010(M) and BBa_K3440009(L)]]
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After insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked colonies corresponding to this part (Figure 1) and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3.
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We then ran electrophoretic gels at 180V for 30 mins (Figure 2) of the products. We obtained two bands (M1 and M5) corresponding to the length of the construct (1288bp).  
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<div><ul>
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<li style="display: inline-block;vertical-align: top;">[[File: T--Stockholm--PlateM.png|thumb|left|300px|Figure 1: Transformation plate of BBa_K3440010 (noted M)]]</li>
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<li style="display: inline-block;vertical-align: top;">[[File:gelML.png|thumb|center|500px|Figure 2: Colony PCR gel for BBa_K34400010(M)]]</li>
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</ul></div>
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 +
 
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M1 and M5 were sent for sequencing to Microsynth AB. The sequence obtained for colony M1 corresponded to the expected part and it  was further analysed by Western Blot (Figure 3) to check whether we obtained protein expression of LuxI thanks to the added myc-tag. As expected, we did not obtain a band neither for uninduced bphR1 nor for bphR1 only induced by PCB and not pbhR2.
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[[File:T--Stockholm--BBa_K3440000_WB.png|thumb|center|500px|Figure 3: Western blot with M1 as BBa_K3440010]]
  
 
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Latest revision as of 23:22, 27 October 2020


luxI-Myc under bphR1 promoter

Pbphr1(BBa_K1155001) - RBS(BBa_B0034) - LuxI(BBa_C0061) - Myc(BBa_K823036)

Usage and Biology

This part expresses LuxI under bphR1 promoter. bphR1 promoter is found in Pseudomonas pseudoalcaligenes KF707 and regulates the transcription of a series of biphenyl catabolic genes. In our project, we rely on this promoter for the detection of polychlorinated biphenyls, pollutants similar to dioxins and found in the Baltic Sea. In particular, in our final system, bphR1 promoter is activated by the complex bphR2:PCB, with bphR2 expressed constitutively and PCB coming from the analysed sea water. LuxI is originally found in Vibrio fischeri (UniProtKB - P12747) and produces 3OC6-HSL, a quorum sensing molecule used in our system to communicate between our detection module in E. coli and our electrical output module in Shewanella oneidensis. In our project, the BBa_K3440010 part is used in order to prove that LuxI can be expressed under bphr1 promoter in the presence of bphR2 and PCBs and to test the leakiness of bphR1 (also tested with BBa_K3440001). A myc tag was added to facilitate characterization.

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 insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked colonies corresponding to this part (Figure 1) and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3. We then ran electrophoretic gels at 180V for 30 mins (Figure 2) of the products. We obtained two bands (M1 and M5) corresponding to the length of the construct (1288bp).

  • Figure 1: Transformation plate of BBa_K3440010 (noted M)
  • Figure 2: Colony PCR gel for BBa_K34400010(M)


M1 and M5 were sent for sequencing to Microsynth AB. The sequence obtained for colony M1 corresponded to the expected part and it was further analysed by Western Blot (Figure 3) to check whether we obtained protein expression of LuxI thanks to the added myc-tag. As expected, we did not obtain a band neither for uninduced bphR1 nor for bphR1 only induced by PCB and not pbhR2.

Figure 3: Western blot with M1 as BBa_K3440010

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 963
    Illegal BglII site found at 1001
    Illegal BamHI site found at 156
    Illegal BamHI site found at 239
    Illegal XhoI site found at 46
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 139