Difference between revisions of "Part:BBa K3440009"
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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 and PCR-amplified them using the VR and VF2 primers contained in the plasmid backbone pSB1C3.
 
After insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked colonies corresponding to this part 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 1) of the products. We obtained two bands, J2 and J3, corresponding to the length of the construct (1299bp).  
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We then ran electrophoretic gels at 180V for 30 mins (Figure 1) of the products. We obtained one band (L3) corresponding to the length of the construct (1433bp).  
  
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[[File:gelML.png|thumb|center|500px|Figure 1: Colony PCR gel for BBa_K3440010(M) and BBa_K3440009(L)]]
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[[File:gelL.png|thumb|center|500px|Figure 1: Colony PCR gel for BBa_K3440009(L)]]
  
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J2 and J3 were sent for sequencing to Microsynth AB. The sequence obtained for colony J3 corresponded to the expected part except for a single mismatch, and it  was further analysed by Western Blot (Figure 3) to check whether we obtained protein expression of RhlR thanks to the added myc-tag. We did not obtain a band of the correct size (27,6 kDa), therefore we could not prove that this construct can express RhlR constitutively.
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L3 was sent for sequencing to Microsynth AB. The sequence obtained for colony L3 corresponded to the expected part and it  was further analysed by Western Blot (Figure 2) to check whether we obtained protein expression of RhlR thanks to the added myc-tag. We did not obtain a band of the correct size (35,1 kDa), therefore we could not prove that this construct can express bphR2 constitutively.
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[[File:T--Stockholm--BBa_K3440000_WB.png|thumb|center|500px|Figure 3: Western blot with J3 as BBa_K3440008]]
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[[File:T--Stockholm--BBa_K3440000_WB.png|thumb|center|500px|Figure 2: Western blot with L3 as BBa_K3440009]]
  
  

Revision as of 16:16, 25 October 2020


bphR2-Myc under Pconst

Pconst(BBa_J23100)-RBS(BBa_B0034)-bphR2 mutated (BBa_K1413021)-Myc (BBa_K823036)-DT(BBa_B0015)

Usage and Biology

This part is used to express bphR2 under a constitutive promoter BBa_J23100 bphR2 gene is originally from Pseudomonas oleovorans produces bphR2, which together with PCB pollutants (polychlorinated biphenyls) can activate bphR1 promoter. In our project, this system is used in our PCB detection module in E. coli. We added a myc-tag to the part in order to be able to characterize it by Western Blotting.

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 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 1) of the products. We obtained one band (L3) corresponding to the length of the construct (1433bp).

Figure 1: Colony PCR gel for BBa_K3440009(L)

L3 was sent for sequencing to Microsynth AB. The sequence obtained for colony L3 corresponded to the expected part and it was further analysed by Western Blot (Figure 2) to check whether we obtained protein expression of RhlR thanks to the added myc-tag. We did not obtain a band of the correct size (35,1 kDa), therefore we could not prove that this construct can express bphR2 constitutively.

Figure 2: Western blot with L3 as BBa_K3440009


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 996
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 536
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 320