Antibiotic resistance poses a significant challenge in the treatment of tuberculosis, requiring the development of new antibiotics with unique molecular mechanisms. In our research, we have identified a specific protein in Mycobacterium tuberculosis as a potential drug target. Let's explore our journey to synthesize and purify this protein.
In order to produce the protein of interest, we employ recombinant DNA technology. This involves isolating and cloning the coding sequence of the protein into an expression plasmid vector. Bacteria such as E. coli are commonly used for this purpose due to their simple manipulation and cost-effectiveness
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
Extraction and purification of product from fermentation is known as Downstream Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of pharmaceuticals product
Cost of the product is determined by the DSP involved
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
Extraction and purification of product from fermentation is known as Downstream Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of pharmaceuticals product
Cost of the product is determined by the DSP involved
Richard's entangled aventures in wonderlandRichard Gill
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Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
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Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
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IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
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Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
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introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
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Purification of protein involved in Mycobacterium tuberculosis as a potential drug target.pptx
1. Addressing
Antibiotic
Resistance in
Treating
Tuberculosis
Antibiotic resistance poses a significant challenge in the
treatment of tuberculosis, requiring the development of new
antibiotics with unique molecular mechanisms. In our
research, we have identified a specific protein in
Mycobacterium tuberculosis as a potential drug target. Let's
explore our journey to synthesize and purify this protein.
by Amit K Singh (2020305001)
2. Recombinant DNA
Technology for Protein
Production
In order to produce the protein of interest, we employ recombinant DNA
technology. This involves isolating and cloning the coding sequence of the protein
into an expression plasmid vector. Bacteria such as E. coli are commonly used
for this purpose due to their simple manipulation and cost-effectiveness.
However, it's important to note that bacterial expression systems lack post-
translational modifications.
3. Introducing Foreign DNA into
E. coli
Transformation is the process of introducing foreign DNA into a cell. E. coli, a
commonly used host, can naturally take up extracellular DNA or be made
artificially competent. Since E. coli is not naturally competent, we induce
competence through electroporation or chemical methods. This allows us to store
and replicate plasmids, which carry the bacterial origin of replication and an
antibiotic resistance gene.
4. Efficient Protein Expression
in E. coli
For the expression of our protein, we turn to E. coli due to its ease of growth and
manipulation, availability of vectors and host strains, as well as the rapid
expression and purification it offers. By overexpressing genes in E. coli BL21
strain, we can achieve large-scale production of recombinant proteins efficiently
and reliably.
5. The Lac Operon and Gene
Transcription
The lac operon found in E. coli plays a crucial role in gene transcription.
Comprising three genes (lacZ, lacY, and lacA), the lac operon is transcribed
together. In the absence of lactose, a repressor binds to the lac operator,
preventing gene transcription. However, in the absence of glucose and presence
of lactose or IPTG, the repressor is released, allowing gene transcription to occur.
6. Purification of the
Protein of Interest
Purification is a vital step in our process. We utilize an
immobilized metal affinity column (IMAC) with Ni2+ - NTA
resin to purify the protein. The protein, fused with an N-
terminal His6-tag, is overexpressed in E. coli BL21(DE3)
cells and harvested. The lysate is then centrifuged, and the
supernatant is applied to the IMAC column. Through this
purification technique, we achieve a highly purified protein
ready for further analysis.
7. Utilizing Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for visualizing and separating protein samples. By running the samples
through a polyacrylamide gel, we can effectively analyze and separate the purified protein. The percentage of polyacrylamide gel used
determines the size of the pores, enabling separation based on protein size. The protein bands can then be detected and analyzed for further
purification if necessary.
8. The Discontinuous Buffer
System in SDS PAGE
SDS PAGE employs a discontinuous buffer system that aids in protein
separation. The running buffer contains Tris, glycine, and SDS at a specific pH.
Tris, with its excellent buffering capacity, is commonly used in SDS-PAGE. The
running buffer's distinct pH and ion composition differentiates it from the gel,
allowing for efficient protein separation and visualization of protein bands.
9. Experimental Procedures:
Cell Culture Growth
The BL21-DE3 strain of E. coli, along with transformed recombinant plasmids,
plays a key role in our experimental procedures. The transformed E. coli are
grown in LB media at 37°C overnight, providing the ideal conditions for growth.
This process facilitates the production of a large amount of mRNA required for
subsequent translation into the targeted recombinant protein.
10. Induction with IPTG
IPTG (isopropyl β-D-1-thiogalactopyranoside) is employed to induce protein
expression in our study. Resembling the structure of lactose, IPTG effectively
triggers the induction process. Once the bacterial culture reaches an optimal
optical density (OD), IPTG is added at a specific concentration. This induction
allows for the translation of the produced mRNA into the targeted recombinant
protein.
11. Cell Lysis and Protein Purification
After induction, the targeted recombinant protein is ready for extraction. The bacterial cells are lysed, leading to the release of the protein of
interest. Once lysed, further purification processes such as centrifugation, chromatography, or other separation techniques can be employed
to isolate and obtain a highly purified form of the protein. The purified protein can then be analyzed using spectroscopic methods for a more
thorough characterization.
12. Conclusion
In conclusion, the study successfully purified and visualized the enzyme associated
with H. pylori using Ni-NTA column chromatography, SDS-PAGE and dialysis. The
purified enzyme was further processed for crystallization.