Members present: Cathy, Celine, Alex, Bohdan, Hamed, Andrea, Davesh, Kat, Tam

Looked into nickel-specific transcriptional regulators

Two candidates were identified: NimR and rcnR (Co²⁺ and Ni²⁺)

Arsenic (non-MerR):

AsrR represses asr via binding, and this binding is inhibited by the presence of Arsenite (see References)

Table of metal regulators (MerR family):

In the article “DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli”:

The article indicates purification protocol suggested and been used for several MerR family in general. It has been suggested to be used for ZntR (Zn(II) homologue in E.coli), CueR (Cu(II) homologue in E.coli) which belong to the MerR family and GolS itself (underneath)

"ZntR gene was cloned from the E. coli chromosome using the primers ZntR N-terminal (59-CTA GTG GAG TAC ATA TGT ATC GCA TTG G-39) and ZntR C-terminal (59-GTA ATC CTG CGG ATC CAA AAA ATC AAC AAC C-39). The 461-bp PCR fragment was digested with NdeI and BamHI and inserted into pET11c (Novagen) digested with the same enzymes. The resulting overexpression plasmid (pET11cZntR) was transformed into BL21(DE3) cells (Novagen). The cells were grown in 9 liters of Luria Bertani broth with shaking at 37 °C and induced with 400 mM IPTG at A600 5 0.6. The cells were harvested by centrifugation 2.5 h after IPTG induction and then stored at -80 °C. The pelleted cells were frozen and thawed three times (45) and then resuspended in 250 ml of Tris buffer A (50 mM Tris-Cl, pH 8.0, 2 mM EDTA, and 5 mM DTT). The cell debris was removed by centrifugation, and the protein in the supernatant was precipitated with 45% (NH4)2SO4. The precipitated protein was then resuspended in 10 ml of Tris buffer A and desalted on a Sephadex G-25 column. The crude protein extract was subsequently loaded onto a Heparin column equilibrated with Tris buffer B (20 mM Tris, pH 8.0, 5 mM DTT). Following elution with a NaCl gradient, the protein fractions were collected and (NH4)2SO4 was added to a final concentration of 1.2 M. The protein was then loaded onto a Phenyl-Sepharose High Performance hydrophobic column (Amersham Pharmacia Biotech) in Tris buffer C (50 mM Tris, pH 8.0, 1.2 M (NH4)2SO4,5mM DTT). The protein was eluted with a decreasing salt gradient, coming off at 0.5–0.3 M (NH4)2SO4, and the protein-containing fractions were concentrated to 2 ml. As a final purification step, the protein was loaded onto a High Load Superdex 75 gel filtration column (Amersham Pharmacia Biotech) equilibrated with Tris buffer D (50 mM Tris, pH 8.0, 250 mM NaCl, 5 mM DTT). The ZntR fractions were concentrated to ;1–2 ml and showed a single band on overloaded SDS-PAGE gels. Protein was stored at 280 °C in Tris buffer D with 5% glycerol. The molecular mass of ZntR (calculated: 16179.3 Da with first Met) was found by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) (PerSpective Biosystems Voyager-DE) to be 16178.1 Da using a sinapinic acid matrix with myoglobin as the calibration standard. "

HiTrap Heparin HP: heparin sepharose (higher cross-linked) 5 of 1mL is $243.04

Supplier: GE Healthcare Life Sciences

Product code: 17-0406-01

Phenyl Sepharose High Performance of 75ml is $513.36

Product code: 17-1082-01

HiLoad Superdex 75 PG is $3098.76

Product code: 28989333

Supplier:Sigma Aldrich

Product code: S100HR-100ML

Sephadex G-25 Medium of 100g is $303.80

Product code: 17-0033-01

Supplier: Sigma Aldrich

Sigma Product code: H6508-5ML

Some of the highlighted parts are significant steps to the procedure and suggested to be costly in the budget. However, based on one article upon CueR purification, they suggested skipping the hydrophobic column step. There are some missing key components on what resin should be used for the heparin column, sephadex

Further contact with Naveen, Kayla, Dr. Thomas O'Halloran and potentially others.

pI of GolS is 9.66 (calculated based on sequence using ExPASy).

We have reassessed an actual 3D structure analysis of GolS, GolS A113T, GolS A113T and P118, CupR.

GolS - Job ID S276431 from I-TASSER:

Visualization of wt GolS with both noncleavable and cleavable His-tags (Ser and Gly ends):

GolS noncleavable His-tag: Tam

GolS cleavable His-tag Gly: Cathy

GolS cleavable His-tag Ser: Alex

Another recommendation upon modeling GolS and its variants:

We will be exploring programs that monitor ligand binding sites particularly different heavy metal ions (Au(III), Au(I), Cu(I), Ag(I), etc...) in order to compare the specificity based on distances of the binding pocket chelation and structural changes between them.

Program suggested at the moment: COACH from University of Michigan

Worked on a protocol from Pardee, K. 2014. Will be posted on benchling when complete

Follow-up text sent to Umar regarding RBS question

- subsection on metal-dependent regulators (nothing on nickel)

-may bind to other metals

-may not be viable, little other information on NimR

-inducing a C35A point mutation in rncR eliminates Co²⁺ binding, making it Ni²⁺ specific

-see figures 2 and 3

-ZntR is a sensitive zinc-specific transcriptional activator

-also binds in small amounts to Cd and Hg

-they made a MerR/ZntR hybrid protein that recognizes Zn with reduced Hg response (see figure 4)

- Compares different exclusion collumns, their resolutions, flow-rate, and ellution efficiency

-Arsenic-sensing/resistance system

-may be overly-complicated

Tasks for tomorrow:

RBS Calculator

Luciferase

i-Tasser with cleavable and non-cleavable His Tags

COACH for ligands

The CRISPR grant

Purification protocol