| New metabolic capabilities are being discovered and connected to underlying genes. New regulation systems, new transport systems, and more information on cellular constituents and cellular processes continue to be found. |
| Scientists use model organisms such as E. coli because they are relatively easy to work with and because there is a vast amount of previous knowledge about them. They can then test whether their findings in this model organism hold true in other species. |
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| Experimentation into details of the biology of E. coli continues unabated,
and the number of papers published annually continues to increase. The number of experimental journal articles on aspects of the basic biology of E. coli has increased from an average of 78 per month in 1996 to an average 94 per month today. New biological information about this well-studied organism continues to roll in. |
 "EcoCyc: a comprehensive database resource for Escherichia coli"
On this site there are many publications about E. coli's genome.
History of E. coli.
Genomic indexing of Salmonella and E.coli |
| Even without knowing everything about it, man has been using |
| genetic knowledge of E. coli for his/her own use. |
| As E. coli can be grown very easily on simple media and its genetic characteristics have been essentially determined, these bacteria are used as vehicles for the preparation of biological polymers, including polypeptide hormones, proteins, carbohydrates, etc. By incorporating the genetic information required to produce such substances into the E. coli genome, it is a simple process to produce these in large amounts.
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| 2. How can we produce new proteins substances by inserting genes to the bacterial genome?
What are the main steps of recombinant proteins production? |
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| To answer those questions, use the information on the following websites.
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| Protein synthesis: Or how to make wonderful proteins in 5 minutes or less:
1) A plasmid from E. coli is removed.
2) A restriction enzyme is used to:
a) cut the DNA of the plasmid at the correct spot (GAATTC).
b) cut out the gene of interest from the human genome.
3) The gene and the plasmid are put together like pieces of a puzzle, and they are "glued" with ligase enzyme.
4) The recombinant plasmid is put back into the E. coli bacterial cell.
5) As the bacterium reproduces millions of times, it copies the new gene too and the new protein is made. |
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| http://biology.technion.ac.il/courses/134065/lectures/lab2006.doc |
| DNA elements directing efficient transcription,
stabilization of the transcript,
powerful translation, resulting in an authentic recombinant protein
and that the protein should stay soluble and accumulate to about 20% of the total cellular protein. |
| The use of E. coli strains as ‘factories’ for protein synthesis is one of the oldest and best-known production techniques and is widely employed in industries throughout the world, due to its easy use, robustness and low cost.
Application of this technology for the production of small molecules began
in 1983. The first product was a strain of E. coli that excreted indigo, one of the oldest known dyes.
More than 20 years have passed since then and regulatory approvals have produced clear guidelines for the production of more recombinant proteins by microbial biotechnology. This is reflected in the sources of products approved for general medical use: |
3. Look at these sites and write down five different substances that are made by E. coli using biochemical know-how.
Write about the uses of those substances |
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DNA probes are used to identify and label DNA fragments that contain a specific sequence. A probe is simply a short length of DNA (20-100 nucleotides long) with a label attached. Two common types of label are used:
· a radioactively labeled probe (synthesized using the isotope 32P) can be visualized with photographic film (an autoradiograph).
· a fluorescently labeled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce in different colors.
Probes are always single-stranded, and can be made of DNA or RNA. If a probe is added to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA containing the complementary sequence. These fragments will now be labeled and will stand out from the rest of the DNA. DNA probes have many uses in genetic engineering:
· To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from a genomic library.
· To identify the short DNA sequences used in DNA fingerprinting.
· To identify genes from one species which are similar to those of another species. Most genes are remarkably similar in sequence from one species to the next, so for example, a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided in the identification of human genes.
· To identify genetic defects. DNA probes have been prepared that match the sequences of many human genetic disease genes, such as muscular dystrophy and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a grid pattern, forming a DNA microarray (or DNA chip). A sample of human DNA is added to the array and any sequences that match any of the various probes will stick to the array and be labeled. This allows rapid testing. |
Scientists are developing new varieties of crops that can withstand harsh growing conditions
Ray Wu, Ph.D., a genetic engineering pioneer and molecular biologist at Cornell University in Ithaca, NY, has added two genes from the E. coli bacterium to rice plants, making them tolerant to drought, cold temperatures and salty soil. The genes produce trehalose, a naturally occurring sugar found in a variety of organisms, including bacteria, that protects them from environmental stresses.
The rice plants grew successfully in the greenhouse and are ready for field testing, says Wu. He predicts that in about five years, the plants can be growing in farmers'
Wu explains that the two E. coli genes in the rice will not harm consumers. "E. coli contains thousands of genes," says Wu. "We are only taking two particular genes that we know will not produce toxic products." As an added precaution, Wu used a "promoter" to control the expression of the E. coli genes: the plant is directed to make trehalose in specific parts, such as the non-edible leaf. Also tested was a second promoter, which serves as an "on-off switch" so that trehalose is made only in response to an environmental stress.
Although rice is a relatively minor crop in the United States, it is a staple in many developing parts of the world, says Wu. And scientists can use the same procedure of inserting genes and a promoter to create biotech wheat, corn and other cereal plants that can withstand harsh conditions. "The world population continues to increase at an explosive rate, our arable land is deteriorating, fresh water is becoming scarce, and increasing environmental stresses will pose ever more serious threats to global agricultural production and food security in future years," says Wu. "Anything we can do to help crop plants cope with environmental stresses will also raise the quality and quantity of food for those who need it most."
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4. If you were to sequence a microorganism's entire genome, in principle, what steps would you take? How would you connect all of the information you obtained, and how would you try to make sense of it (for example, find out where the genes are and what they are likely to code for)? |
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5. One of the challenges of combinatorial mutagenesis is the selection of molecules with desired properties. This is particularly problematic if degeneracy has been introduced at a large number of codons simultaneously, resulting in a very low expected frequency of those molecules. Can you think of procedures you could use (for example, addition of extra steps) to boost the frequency of the desired molecules? |
| Genomic indexing of Salmonella and E.coli |
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