This document summarizes experiments to locate regions in the bacterial protein DnaK that are important for its chaperone activity. DnaK is a molecular chaperone found in E. coli and similar proteins exist across organisms. It aids protein folding and protects cells from stress. The structure of DnaK and a peptide-binding mutant are described. Methods are outlined to mutate plasmids, overproduce proteins, and purify DnaK using chromatography. Future plans involve measuring chaperone activity by monitoring refolding of denatured green fluorescent protein. The significance is that inhibiting chaperones may help cure diseases caused by protein misfolding such as cancer and cystic fibrosis.
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The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues.
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clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria. Now CRISPR use as genome editing tool in different Plant Breeder to manipulate the DNA of the crop
“Gene drives,” a technology for controlling genetic traits, could revolutionize disease prevention. But nature has a way of thwarting scientific meddling.
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https://www.creative-biolabs.com/gene-therapy/approaches-to-genome-editing.htm
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Genome Editing Techniques by Kainat RamzanKainatRamzan3
Genome technology has revolutionized biological science through techniques of Gene Editing in order to edit any organism's genome.MegNs and zinc-finger nucleases are commonly understood to be used, as is the effector's transcriptional activator-like nucleases. In CRISPR/Cas9, genetic alterations, and gene functionality have become a well-known tool for understanding gene targeting.
Gene editing application for cancer therapeuticsNur Farrah Dini
The application of TALENs as one of the gene editing tools in order to modify a specific targeted sites on a genome. This method shows a tremendous benefits especially in cancer research.
Genome editing is a method of making specific changes to the DNA of a cell or organism. An enzyme scissors the DNA at a specific sequence, and when this is repaired by the cell, a change or ‘edit’ is made to the sequence.
https://www.creative-biolabs.com/gene-therapy/approaches-to-genome-editing.htm
Crispr-Cas9 system works on the concept of bacterial defence mechanism. The idea of which was replicated in eukaryotic cell in in- vitro condition by the researchers.
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1. Locating regions in Dnak
important for chaperone activity
Roxana Hernandez
7/27/12
DNA Molecular Laboratory, NCI,NIH
2. DnaK
• Source from Escherichia
coli
• DnaK/Hsp70 (70
kilodalton heat shock
proteins) with similar
structure exist in almost
all living organisms
• Important for protein
folding and to help
protect cells from stress
• Functions with co-
chaperones
6. Methods
Mutated plasmids transformed into cells
for growth using ‘QuickChange’ for Site-
directed mutagenesis
Collected DnaK 351 mutated plasmid for
sequencing
If mutation is correct then the plasmid is
used for protein overproduction
7. Methods
• Protein prep:
① Grow culture
(overproduction of
proteins)
② Lyse cells using a
French Press
③ Column
Chromatography
kDa
181.8
82.2
64.2
48.8
37.1
25.9
19.4
M P S P S U I
14.8
10. Future Plans
Monitor disaggregation by
measuring the increase in
fluorescence
Native Green
Fluorescent Protein
(GFP)
Heat
denature
Incubate with chaperones
& ATP
Refolded
GFP
Heat-aggregated
non-fluorescent GFP
11. Significance
• Investigating the role of chaperones in
diseases of protein misfolding
• Inhibiting the chaperone involved could
help cure disease
– Ex: Cancer, Cystic Fibrosis, Sickle Cell, Type II
Diabetes..
12. Acknowledgements
• Sue Wickner
• Shannon Doyle
• Joel Hoskins
• Danielle Johnston
• Olivier Genest
• Colleen Berringer
DNA Molecular Laboratory, NCI,NIH
Editor's Notes
Dnak 351 mutant on an overproduction plasmid
Site-directed mutagensis
Move mutated plasmids into cells and grow
Plasmid prep—collect mutated plasmid
Sequence mutated plasmid
If sequence has correct mutation move into cells for protein production
Grow large culture (overproduce protein)
Lyse cells using french press
Separate proteins from cell debris (pellet form)
Anonic exchange column (Q-sepharose column)
Gel filtration (S100 Column) size exclusion
M-Marker
P-Pellet
S-Supernatant
U-Uninduced
I-induced
You should do an arrow that goes from the Q18 in Q-seph and Brackets to the Sfractions
**Gels from Columns S100 & Q-sepharose
Anion Exchange= Q-Sepharose Columns
Size Exclusion= S100 Columns
D=dnaK Protein Supernatant
M=Marker
kDa
Supernatant is tested for the presence of the target protein and checked for purity
**S14 should have been a little more than that it fell out