IGEM:Brown/2007/Sensor/What to detect?
Bacterial signal transduction network in a genomic perspective
author: Michael Y. Galperin
Tables 1 and 2 show what types of signalling molecules are present in different types of prokaryotes.
It looks like S_TKc (Serine-Threonine kinase, catalytic) would work best for E. Coli because there are not many of them in E. Coli naturally. Or, we may want to use a signalling protein that doesn't exist yet in E. Coli, to prevent confusion and false signals.
Signal transduction: Hair brains in bacterial chemotaxis
authors: Jeff Stock and Mikhail Levit
In their ‘ON’ state, in the absence of attractants, several receptors bind to CheA in such a way that CheA is activated over 100-fold [24], and conversely CheA binding to the receptors appears to be required for long-range structural interactions that serve to organize the array. In the ‘OFF’ state, in the presence of attractants, it is as if the CheA dimer is torn apart by binding to the receptor network.
In E. coli, at least five different receptors — Tar, Tsr, Tap, Trg and Aer — appear to be intermingled within the same complex.
a) Tar: aspartate and maltose; cobalt and nickel
b) Tsr: serine
c) Tap: Taxis towards peptides
d) Trg: ribose and galactose
e) Aer: directs taxis towards ribose, galactose, maltose, malate, proline and alanine
a. CheA is a kinase that takes a phosphate off of ATP and attaches it to itself
b. CheW connects CheA to the chemoreceptor
Binding Proteins
author: Dr. Leonidas G. Bachas
Sensing System for Zinc Based on Zinc-Binding Protein
Zinc is an essential element in our diet. Too little zinc can cause problems, but too much zinc is also harmful. Severe soil zinc deficiency can cause complete crop failure. Certain microorganisms are known to survive in highly toxic environments contaminated with toxic species such as mercury, arsenic, cadmium, zinc, lead, copper or nickel. Resistance is associated with presence of resistance operons which are precisely regulated. Operon consists of gene for regulatory protein to which toxic metals binds and induce the expression of other genes of operon. Detoxification occurs either by pumping the toxic metals out of the cell or by expression of metallothionein, a cysteine rich protein that chelates heavy metals. We plan to take advantage of this specific binding between the regulatory protein and the toxic species in order to develop a sensing system for the target toxic analyte. In this project we have replaced the genes of the operon for zinc resistance with the genes that encode for reporter proteins to develop a biosensor for zinc.
Detection of Sulfate Using Periplasmic Sulfate-Binding Protein
Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe, which allow the development of reagentless sensing systems for their corresponding ligands. On the basis of this strategy we have evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe for rational designe of a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. Different environment-sensitive fluorescent probes were then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.
An Exceptionally Selective Lead(ii)-Regulatory Protein from Ralstonia Metallidurans: Development of a Fluorescent Lead(ii) Probe
Lead Detection Paper (THIS IS AWESOME!)
PbrR protein [Ralstonia metallidurans CH34]
Other Aliases: pMOL30_092
Genomic context: Plasmid pMOL30 (Plasmid 1)
Annotation: NC_006466.1 (114932..115370)
GeneID: 3170418
MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLS
YRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTT
AHPSD
114933..115370 (including stop codon)
DNA sequence:
atgaatat ccagatcggc gagcttgcca
agcgcaccgc atgcccggtg gtgaccattc gcttctacga acaagaaggg ctgttgccgc cgccgggccg cagccggggg aattttcgcc tgtatggcga ggagcacgtg gagcgcttgc agttcattcg tcactgccgg tctctggata tgccgttgag cgacgtacgg accttattga gttaccggaa gcggcccgac caggattgcg gtgaagtcaa tatgctcttg gatgagcaca tccgtcaggt cgaatctcgg atcggagctt tgctcgaact gaagcaccat ttggtggaac tgcgcgaagc ctgttctggt gccaggcccg cccaatcgtg cgggattctg cagggactgt cggactgcgt gtgtgatacg cgggggacca ccgcccatcc aagcgactag
