Difference between revisions of "Part:BBa K3440013"
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<partinfo>BBa_K3440013 short</partinfo> | <partinfo>BBa_K3440013 short</partinfo> | ||
| − | Pconst(BBa_J23100)-RBS(BBa_B0034)-LuxR(BBa_C0062)-Myc(BBa_K823036) | + | Pconst(BBa_J23100) - RBS(BBa_B0034) - LuxR(BBa_C0062) - Myc(BBa_K823036) |
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This part constitutively produces LuxR. | + | |
| + | This part constitutively produces LuxR. LuxR, originally expressed in Vibrio Fischeri (UniProtKB - P12746), is part of a quorum sensing system with LuxI. When LuxI is expressed, it produces a signalling lactone 3OC6-HSL which can bind to LuxR and activate the Plux promoter. This system is used to create communication between our E. coli detection module and our electrical output circuit in Shewanella oneidensis. | ||
| + | In our project, the BBa_K3440013 part was used to prove that LuxR could be produced under a constitutive promoter in E. coli TOP10 cells. | ||
===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. | ||
| + | After insertion into a pSB1C3 plasmid and transformation of E. coli TOP10 cells using heat shock, we picked several colonies (Figure 1) 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 2). In particular, we obtained P6 for which the band height corresponded to the expected length of the construct (1328 bp). | ||
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| + | <div><ul> | ||
| + | <li style="display: inline-block;vertical-align: top;">[[File: T--Stockholm--PlateP.png|thumb|left|300px|Figure 1: Transformation plate of BBa_K3440013 (noted P)]]</li> | ||
| + | <li style="display: inline-block;vertical-align: top;">[[File:T--Stockholm--gelNOP.png|thumb|center|500px|Figure 2: Colony PCR Gel for BBa_K34440011(N), BBa_K3440012(O) and BBa_K3440013(P)]]</li> | ||
| + | </ul></div> | ||
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| + | We sent P6 for sequencing to Microsynth AG, but the Sanger sequencing revealed that the amplified product was not the expected one. We therefore highly recommend other teams to always send their samples for sequencing, even when the bands on gels have the right size | ||
Latest revision as of 23:29, 27 October 2020
LuxR-Myc under constitutive promoter
Pconst(BBa_J23100) - RBS(BBa_B0034) - LuxR(BBa_C0062) - Myc(BBa_K823036)
Usage and Biology
This part constitutively produces LuxR. LuxR, originally expressed in Vibrio Fischeri (UniProtKB - P12746), is part of a quorum sensing system with LuxI. When LuxI is expressed, it produces a signalling lactone 3OC6-HSL which can bind to LuxR and activate the Plux promoter. This system is used to create communication between our E. coli detection module and our electrical output circuit in Shewanella oneidensis. In our project, the BBa_K3440013 part was used to prove that LuxR could be produced under a constitutive promoter in E. coli TOP10 cells.
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 several colonies (Figure 1) 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 2). In particular, we obtained P6 for which the band height corresponded to the expected length of the construct (1328 bp).
We sent P6 for sequencing to Microsynth AG, but the Sanger sequencing revealed that the amplified product was not the expected one. We therefore highly recommend other teams to always send their samples for sequencing, even when the bands on gels have the right size