Team:UTK-Knoxville/construct

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Team:UK-Knoxville - 2013.igem.org


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Construst

Buffers & Solution

COMING SOON

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Kits

Coming Soon

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Marker

COMING SOON

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Enzymes

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    • Cytoplasmic foundation

      • TodS?

    • Protein design

      • Aer - Aer is an aerotaxis sensing flavoprotein in E. coli. It is a member of the superfamily of PAS domain proteins, a group of which sense oxygen, redox potential and light. Aer is an intracellular sensor that is bound to the membrane, so it mediates aerotactic responses based on the oxygen and energy state of the cell.

Design: For our chimera of Aer and EnvZ, we used the sensing domain of Aer, through its transmembrane regions, and up to its HAMP domain, since the HAMP and sensor domains work together for signal transduction. The histidine kinase region of EnvZ was used and the two pieces were fused together.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2007.05889.x/full#b4



      • NarX - NarX is a transmembrane nitrate/nitrite sensor from E. coli.      Michael Manson et al. [Source] have already fused the Tar sensor to the NarX histidine kinase domain with good results.  Our design attaches the NarX sensing domain to the EnvZ HAMP and kinase domains.

        • Scott M. Ward, Arjan F. Bormans, and Michael D. Manson. Mutationally Altered Signal Output in the Nart (NarX-Tar) Hybrid Chemoreceptor. Journal of Bacteriology. 188 (2006): 3944-3951 doi:10.1128/JB.00117-06.

      • HemAT - HemAT senses oxygen from Bacillus subtilis.  HemAT is a cytoplasmic sensor, so we have attached it to the TodT reporter machinery.

Fig. 4. The schematic models of (A) the sensor domain of HemAT-Bs and (B) the sensor and transmembrane domains of MCP. The crystal structures of sensor domains in HemAT-Bs (PDB: 1OR6) and Tar from Salmonella typhimurium (PDB: 1WAT) that is a typical MCP are shown in (C) and (D), respectively. (E) and (F) are the top view of (C) and (D), respectively. The helices colored in red are the constituent of the conserved helix bundle.

Figure 1

Figure 1. The Molecular Structure of the HemAT Sensor Domain Represented with Ribbon Diagrams(A) Stereo view of the structure. The signaling domain would be located further down on the page.(B) Top view showing the flanking of the core helices, G and H, by the rest of the molecule. The helices are labeled corresponding to the nomenclature of the globin fold. Subunit A, cyan; subunit B, yellow.

      • TodS - TodS primarily senses toluene but also senses benzene and styrene.  We are attaching the TodS sensing domain to EnvZ because TodS is cytoplasmic and we want to see if a cytoplasmic sensor works with a transmembrane histidine kinase domain.  TodT is also our chosen platform for cytoplasmic sensor domains.

      • RcoM -

      • LasR -

    • Plasmids

 

 

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Name Sequence

Melting Temperature

(°C)

Function / Comment Status
General (Transmembrane)        
EnvZ_r aaaaaactcgagttaggatccTTACCCTTCTTTTGTCGTGCC 57 Lowercase part has restriction sites: XhoI-ctcgag BamHI-ggatcc In
OmpR_r AAAAAACTCGAGAGGGGATCtcatgctttagagccgtccgg 60 Restriction sites: XhoI-ctcgag BamHI-ggatcc In
OmpR_f aaaaaaCATATGCAAGAGACCTACAAGATTCTG 53 Restriction site: NdeI-catatg In
Aer        
Aer_f aaaaaagaattctaaagatctaaggagatataATGTCTTCTCATCCGTATGTC 53 Lowercase part has restriction sites: EcoRI-gaattc BglII-agatct In 5/30
Aer-EnvZ_1r GCGCAAGTCGTGACTTACCCatttctgacactggacacctgg 58 Overlapping PCR primers In 5/30
Aer-EnvZ_1f ccaggtgtccagtgtcagaaatGGGTAAGTCACGACTTGCGC 59 Overlapping PCR primers In 5/30
NarX        
NarX_F        
NarX_R        
NarX-EnvZ_R        
HemAT        
HemAT_F aaaaaactcgagaaggagatatattgttatttaaaaaagacagaaaacaag 52 XhoI- ctcgag  
HemAT_R ggcgctggttatccatcggactggttgtactcgctttgaaacg 58 Overlapping PCR primers  
HemAT_NtrB_F cgtttcaaagcgagtacaaccagtccgatggataaccagcgcc 61 Overlapping PCR primers  
TodS        
         
         
         
RcoM        
RcoM_F aaaaaactcgagaaggagatataTTAGTTTTTACTTGCTTCAGTTGG 54 XhoI-ctcgag  
RcoM_R ggcgctggttatccatcggaatgtttaaatattttgcaattgcttc 52 Overlapping PCR primers  
RcoM_NtrB_F gaagcaattgcaaaatatttaaacattccgatggataaccagcgcc 61 Overlapping PCR primers  
LasR        
         
         
         
Cytoplasmic Construct        
NtrC_F aaaaaacatatgatgcaacgagggatagtctgg 58 Restriction site: NdeI-catatg  
NtrC_R agatccgagctggggatggagtgactcgagaaaaaa 57 XhoI-ctcgag  
NtrB_R aaaaaactcgagttaggatccTTATTTCCTGATAGGCAGGTAAAC 54 Lowercase part has restriction sites: XhoI-ctcgag BamHI-ggatcc  
         
         
         
         
         
         
         
         

 

Synthetic oligonucleotides

 

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Phages

Name application reference
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Bacteria

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Bacteria Growth Media

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UTK.edu UTK CBE UTK CBE Trinh Lab