Team:Pittsburgh/Parts
Parts
We have purchased nine plasmids from Addgene to serve as our temperature sensor templates and further characterized their activities using E. Coli BL21 cells. The temperature sensor plasmids generally contain a regulator element as well as the sequence which can express mWasabi, a green fluorescent protein. pTlpA-Wasabi-NF serves as the control plasmid since it codes for a non-fluorescent mWasabi. Overall, the plasmids allow mWasabi expression at elevated temperatures. In the BL21 transformed with TlpA and TcI plasmids, high temperature causes the transcription repressors to un-dimerize and allows transcription to occur. In the BL21 transformed cells with heat shock promoter plasmids, high temperature engages endogenous transcription factors to induce transcription.
We also implemented plasmids with constitutive RFP expression (mTangerine) to test the relative mWasabi expression levels. For these constructs, each cell will both constitutively express mTangerine and mWasabi in response to thermal induction. This allows us to measure levels of fluorescence using a plate reader, microscopy, flow cytometry.
We tested with the BBa_J36848 as a way to attach magnetic nanoparticles to the surface of the bacteria through biotin-streptavidin interaction.
Composite Parts
| Construct | Output Gene Products | Source | Part number |
|---|---|---|---|
| pTlpA-mWasabi | TlpA, Wasabi | S. Typhimurium, Clavularia | Bba_K3666102 |
| pTlpA-mWasabi-NF | TlpA, Nonfluorescent Wasabi | S. Typhimurium, Clavularia | Bba_K3666103 |
| pTcI-mWasabi | TcI, Wasabi | Bacteriophage Lambda, Clavularia | Bba_K3666104 |
| pDnaK-mWasabi | Wasabi | Clavularia | Bba_K3666105 |
| pRpoH-mWasabi | Wasabi | Clavularia | Bba_K3666106 |
| pLaqI-mWasabi | Wasabi | Clavularia | Bba_K3666107 |
| pClp-mWasabi | Wasabi | Clavularia | Bba_K3666108 |
| PHtpG-mWasabi | Wasabi | Clavularia | Bba_K3666109 |
| PGrpE-mWasabi | Wasabi | Clavularia | Bba_K3666110 |
| PGrpE-mWasabi | Wasabi, Tangerine | Clavularia | Bba_K3666110 |
| pTlpA-mWasabi-mTangerine | Tlpa, Wasabi, Tangerine | S. Typhimurium, Clavularia, Discosoma | Bba_K3666111 |
| pTlpA-mWasabi-NF-mTangerine | TlpA, Nonfluorescent Wasabi | S. Typhimurium, Clavularia, Discosoma | Bba_K3666112 |
| pTcI-mWasabi-mTangerine | TcI, Wasabi, Tangerine | Bacteriophage Lambda, Clavularia,Discosoma | Bba_K3666113 |
| pDnaK-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3666117 |
| pRpoH-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3666114 | pClp-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3666115 |
| PHtpG-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3666116 |
| PGrpE-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3866118 |
| pLacIq-mWasabi-mTangerine | Wasabi, Tangerine | Clavularia, Discosoma | Bba_K3666119 |
Basic Parts
| Construct | Classification | Source | Part number |
|---|---|---|---|
| TlpA promoter | Regulatory | TlpA comes from S. Typhimurium | Bba_K3666000 |
| pR | Regulatory | Bacteriophage Lambda | Bba_k3666001 |
| pL | Regulatory | Bacteriophage Lambda | Bba_K3666002 |
| pRpoH promoter | Regulatory | | Bba_K3666003 |
| pClpP/ClpX | Regulatory | | Bba_K3666004 |
| pHtpG promoter | Regulatory | | Bba_K3666005 |
| pDnaK promoter | Regulatory | | Bba_K3666006 |
| pGrpE promoter | Regulatory | | Bba_K3666007 |
| lacIq promoter | Regulatory | | Bba_K3666008 |
| p14 | Regulatory | | Bba_K3666013 |
| mWasabi | Reporter | Clavularia | Bba_k3666009 |
| mWasabi-NF | Reporter | Clavularia | Bba_k3666010 |
| mTangerine | Reporter | | Bba_k3666011 |
| sfGFP | Reporter | | Bba_k3666012 |
| TlpA | Transcription repressor | S. Typhimurium | Bba_K3666015 |
| Tcl | Transcription repressor | Bacteriophage Lambda | Bba_K3666016 |