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Revision as of 06:16, 19 October 2016

console.js:26

Week 3: June 1 2016

Wednesday, 6/1
Members present: Hamed, Kat, Tam, DK, Cathy
Lab Stuff: (research, plasmid design, protocol design, troubleshooting)
Plasmid design:
●
Created plasmid constructs of the following inserts. Note that the sequences have yet to be codon optimized and therefore the actual sequences are subject to change
Reporter Constructs
GFP reporter plasmid v1.png
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TetO promoter and GFP reporter plasmid, in pSB1C3 backbone. v1
TetO promoter and mCherry Reporter Plasmid, in the pSB1C3 backbone. v1
mCherry reporter plasmid.png
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large plasmid reporter (GFP and mCherry).png
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Large reporter plasmid, with both GFP and mCherry, all in the pSB1C3 backbone. v1
GolS Constructs
Small Plasmid GolS_tetO - GolS.png
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Small plasmid of GolS with the constitutive TetO promoter, all in the pSB1C3 backbone. v1
Small plasmid of lacZ-alpha with the pgolB promoter, all in the pSB1C3 backbone. v1.
Small plasmid GolS _pgolB - LacZ-alpha.png
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Large plasmid GolS (P118A) v1.png
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Large plasmid of GolS with the TetO promoter, and of lacZ-alpha with the pgolB promoter, in the pSB1C3. v1.
GolS (P118A) Constructs
Small plasmid GolS (P118A) _TetO-GolS (P118A).png
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Small plasmid of GolS (p118A)with the constitutive TetO promoter, in the pSB1C3 backbone. v1
Large plasmid of GolS (p118A)with the TetO promoter, and of lacZ-alpha with the pgolB promoter, in the pSB1C3 backbone. v1.
Large plasmid GolS (P118A) v1.png
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rcnR constructs
Small plasmid rcnR (C35A)_tetO - rcnR (C45A).png
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Small plasmid of rcnR (C35A), along with a constitutive TetO promoter, all in the pSB1C3 backbone. v1.
Small plasmid of LacZ-alpha along with the prcnA promoter, in the pSB1C3 backbone. v1.
Small plasmid rcnR (C35A)_prcnA - LacZ-alpha v1.png
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Large Plasmid rcnR (C35A) v1.png
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Large plasmid, with rncnR and the TetO promoter, along with LacZ-alpha and the prcnA promoter, all in the pSB1C3 backbone, v1
UNS Bridging Oligos
A
B
C
D
1
Oligo BridgeMelting TempOligo SequenceLength (bps)
2
UNS1 - UNS473.4CCATTACTCGCATCCATTCTCAGGCTGTCTCGCTGACCTCCTGCCAGCAATAGTAAGACAACA62
3
UNS5 - UNS672.5CGAGCCAACTCCCTTTACAACCTCACTCAAGTCTCGTTCGCTGCCACCTAAGAATACTCTACG62
4
UNS8 - UNS973.7CCCTCGTCTCAACCAAAGCAATCAACCCATCGTTCCTTATCATCTGGCGAATCGGACCCAC60
5
UNS4 - UNS672.5CCTGACCTCCTGCCAGCAATAGTAAGACAACACTCGTTCGCTGCCACCTAAGAATACTCTACG62
6
UNS5 - UNS973.1CGAGCCAACTCCCTTTACAACCTCACTCAAGTGTTCCTTATCATCTGGCGAATCGGACCCAC61
Table1
Input to I-TASSER structure of cleaved His-tag (Ser & Gly versions) GolS:
Job ID: S277159 - GolS cleaved His (Gly) - Kat
Job ID: Unknown - GolS cleaved HIs (Ser) - Cathy
His-tag presence apparently alters the structure of the GolS and its binding site by a small margin.
Exploring protein-ligand binding program:
SWISSDOCK: ongoing - attempt to insert gold ions into the system.
However, it appears that metal ligand binding site cannot be monitored using a monomer of GolS alone. Chances are in order to fully simulate metal ligand binding site, it might require a mono-dimer structure of GolS to come together.
COTH: dimerization of GolS prediction program is the highest priority.
Job ID: S9165A-S9165B - wt GolS - Cathy
Job ID: unknown - uncleavable His-tag GolS - Tam
Problems with COTH:
ID: S9165A-S9165B
The result is complete. 3D visualization uses .pdb files with only point forms that does not give a great depiction of the homodimer structure.
GolS predicted dimer structure.PNG
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This is not sufficient enough to visualize dimerization of the wt GolS. We suspect that COTH is using homology to predict dimerization.
COACH:
Job ID: CH034136
GolS ligand binding prediction is complete. The program shows potential binding sites of GolS for DNA duplex and metal ions which suggested to be Zn(II) and Cu(I).
DNA duplex:
GolS binding to DNA at N-term.PNG
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There is a clear indication that GolS N-terminus is greatly involved in the DNA-binding process. His-tag at N-terminus will not work as it will alter the binding affinity of GolS to DNA (which is an important function of GolS).
Metal Ions:
GolS binding to metal ions (Zn for ex)..PNG
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GolS is also suggested to bind to metal ions at the helix-turn-helix loop which was suggested by the model to chelate any metal ions. The COACH model suggested Zn(II), Cu(I). However, we can pre-assume that Au(III), Au(I) are potential ligand (Au ions are not in the database).
Re-analysis of CueR homologue:
CueR binding to Au(I).PNG
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The list of coordinated residues involved in chelation of Au(I) for CueR structure is shown below.
A
B
C
D
1
CoordinationDistance (A)DIstance RMSDBest geometry
2
#1 CYS 112.B SG2.290N/A
3
#1 CYS 120.B SG2.390.043octahedron
4
#1 SER 77.A O3.430.088cube
Table2
CueR vs GolS aa sequence alignment.PNG
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GolS (Model 3) sequence and CueR (the other), despite some differences, show similar residues at the binding site for metal ions (res 112, 120 and 122). Notice that Ile122 (hydrophobic molecule) does not play a major role in the structure of the molecule.
Administrative: (shipment orders, inventory)
Email Updates: (Correspondence with professors, shipping companies, etc)
●
Sent email to Dr. Papangelakis (subject: BioZone Collaboration), requesting a meeting to advice on expanding biosensing to other metals and using gold chloride in the lab
●
Sent draft plasmids to Kayla and Naveen. We also asked them which codon optimization tool would be most appropriate.
Meeting Notes:
References: