Team:UTK-Knoxville/construct

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Contents [hide] 1 Constructs 1.1 Buffers & Solution 1.2 Kits 1.3 Marker 1.4 Enzymes 1.5 Plasmidvectors 1.6 Synthetic oligonucleotides 1.7 Phages 1.8 Bacteria 1.9 Bacteria Growth Media

 

 

 

 

Construst

Buffers & Solution

COMING SOON

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Kits

Coming Soon

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Marker

COMING SOON

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Enzymes

COMING SOON

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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. 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

 

COMING SOON

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Phages

Name	application	reference
COMING SOON

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Bacteria

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Bacteria Growth Media

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