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Genetic Engineering

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The use of bacterial and microbial genetics to isolate, manipulate, recombine, and express genes

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Genetically Modified Organisms (GMOs)

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An organism produced by genetic engineering

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Genomics

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The study of an organism’s gene structure and gene function in viruses and organisms

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Genetic Recombination

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The process of bring together different segments of DNA; A natural mechanism for DNA transfer from one microorganism to another

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Vertical Gene Transfer (VGT)

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the passing of genes from one cell generation to the next; When a mutation occurs in the parent cell, all future generations derived by binary fission from the parent cell will have the same mutation

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Horizontal Gene Transfer (HGT)

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The movement of genes from one organism to another within the same generation; a type of genetic recombination that involves the lateral intercellular transfer of DNA from a donor cell to a recipient cell

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Horizontal Gene Transfer (HGT)

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If the recipient cell receives from the donor a chromosomal DNA fragment, a plasmid, a transposon, or a combination of all of them containing a gene for antibiotic resistance, the new DNA pairs with a complementary region of recipient DNA and replaces it. This is an example of?

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1.) transformation 2.) conjugation 3.) transduction

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Three Distinctive Mechanisms that mediate HGT b/w bacterial cells

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1.) Readied for transfer
2.) Transferred to the recipient cell
3.) Taken up successfully by the recipient cell
4.) Incorporated in a stable state in the recipient

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Four Steps of HGT mediation mechanisms

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Transformation

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the transfer and integration of DNA fragment from a dead and lysed donor cells to a recipient cell’s chromosome

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Transformation

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the conversion of a normal cell into a malignant cell due to the action of a carcinogen or virus; the uptake of a free DNA fragment from the surrounding environment and the expression of the genetic information in the recipient cell, by integration of the DNA fragment, the recipient (transformant) has gained some ability it previously lacked

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Transformation

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S. pneumoniae species that causes pneumonia has 2 different strains: a wild-type encapsulated strain, S, because it grows in smooth colonies, and an unencapsulated mutant strain designed R because it s rough and harmless. This is an example of?

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Competence

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referring to the ability of a cell to take up naked DNA from the environment; usually incorporates only one or at most a few DNA fragments

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Transformation

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in natural environments this process occurs, when bacterial cells die and lyse, the chromosome typically breaks apart into fragments of DNA composed of about 10-20 genes. Uptake of DNA fragments (or a plasmid) by recipient cells appears to occur only at the cell poles, as this is where the competence factors are found and actual DNA uptake has been observed.

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Internalization of DNA

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an ATP dependent process and requires DNA binding proteins, cell wall degradation proteins, and cell membrane transport proteins

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Gram Positive Internalization of DNA

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Bacillus subtilis and S. pneumoniae degrade one single stranded strand of DNA fragment as it is being taken into the cell, and will replace a similar chromosome sequence. This is example of

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Gram Negative Internalization of DNA

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H. influenzae a double stranded DNA fragment is transported into the periplasmic space, but then one strand is digested by a nuclease before transport into the bacterial cytoplasm. This is example of what?

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Transformation

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an organism’s ability to cause disease is increased either by acquiring genes from another more lethal organism or two mildly pathogenic organisms cumulate and the recipient becomes more dangerous or have drug resistance by acquiring R plasmids. This is a result of what?

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Conjugation

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In Bacteria and Archaea, a unidirectional transfer of genetic material from a live donor cell into a live recipient cell during a period of cell contact; two live bacterial cells come together and the donor cell directly transfers DNA to the recipient cell

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Conjugation Pilus

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A protein filament essential for conjugation b/w donor and recipient bacterial cells

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The donor cell F+ (because it contains the F Factor) and it contacts the recipient cell F- (because it lacks the F Factor)

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For cell to cell contact, what cell produces the conjugation pilus? Why?

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F Factor

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A plasmid containing genes for plasmid replication and conjugation pilus formation

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Rolling-Circle Mechanism

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A type of DNA replication in which a strand of DNA “rolls off” the loop and serves as a template for the synthesis of complementary strand of DNA; takes about 5 minutes

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Origin of Transfer (OriT)

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The fixed point on an F plasmid (factor) where one strand is nicked and transferred to a recipient cell

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Horizontal Gene Transfer

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DNA synthesis in the donor cell produces a new complementary strand to replace the transferred strand, once the DNA transfer is complete, two cells separate

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Horizontal Gene Transfer

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In the recipient cell, the new single stranded DNA serves as a template for synthesis of a complementary polynucleotide strand, which ten circularizes to reform an F factor, which completes the F- to F+ conversion and is now capable of conjugating with another F- recipient (only acquires genes on the F factor)

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Horizontal Gene Transfer

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It is a highly efficient form of DNA transfer, that spreads rapidly, converting a whole population into plasmid containing cells, and it is the major mechanism for antibiotic resistance transfer

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High Frequency of Recombination (Hfr)

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referring to a bacterial cell containing an F factor incorporated into the bacterial chromosome

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Hfr strains

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the F factor has attached to the chromosome, this attachment is a rare event requiring an insertion sequence to recognize the F factor

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High Frequency of Recombination (Hfr) Conjugation that Transfers Chromosomal DNA

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Once incorporated into the chromosomal DNA, the F Factor no longer controls its own replication, but the Hfr cells trigger conjugation just like an F+ cell. When a recipient cell is present, a conjugation pilus forms and attaches to the F- cell. Then the two cells are brought together. One strand of the donor chromosome is nicked at oriT and a portion of the single-stranded chromosomal/plasmid DNA then passes into the recipient cell

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OriT site

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What is the site called in the chromosome in the middle of the F Plasmid gene?

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The first genes to enter are only a part of the F factor and they do not make it an F+ donor cell, but the last genes are, and they rarely enter the recipient cell. Conjugation is interrupted by movements that break the bridge b/w cells before complete transfer is finished

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The OriT site in the chromosome is in the middle of the F plasmid gene, so in the recipient cell, How do genes enter the cell? Why do these recipient cells never become F+ donors? how is conjugation is usually interrupted?

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Recombinant F- cell

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that received a few chromosomal genes and partial F factor genes from a donor cell during conjugation

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The F factor usually detaches from the chromosome, and the enzymes synthesize a strand of complementary DNA. The F Factor forms a loop to assume an existence as a plasmid to make the recipient an F+ donor cell

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If the entire chromosome is transferred to the recipient, what happens to the F factor? How does the recipient become an F+ donor cell?

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F' Plasmid

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An F plasmid carrying a bacterial chromosome fragment

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F' plasmid
the recipient cell is partially a diploid organisms because they have 2 genes for a gene function

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When it is transferred in subsequent conjugation, the recipient recieves the chromosomal genes excised from donor and the genes from the plasmid DNA, which means it has 2 pairs of genes for the same process. What does this happen to? What does this make the recipient cell?

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It occurs in an Hfr cell
an integrated F plasmid

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In this cell, ____breaks free from the chromosome and in the process takes along a fragment of chromosomal DNA. Name the cell this occurs to and what the ___ is.

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Gram Negative

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Conjugation occurs b/w cells of various bacterial genera; e.g.like Escherichia and Shigella, Salmonella and Serratia, Escherichia and Salmonella. What are these cells? Gram Negative or Positive?

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yes, but they do not use conjugation pili

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Are gram positive microbes capable of conjugation? if so, does it involve conjugation pili?

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Gram Positive Conjugation

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it only involves plasmids carrying genes for antibiotic resistance and there are no pili, but the recipient cell secretes substances encouraging the donor cell to produce clumping factors composed of proteins, which causes the factors to come together (clump) the donor and recipient cells, and pores form between the cells to permit the plasmid transfer

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Transduction

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The transfer of a few bacterial genes from a donor cell to a recipient cell via a bacterial virus

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Bacteriophage/phage

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A virus that infects an replicates within bacterial cells

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Lytic Cycle

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A process by which a bacterial virus replicates within a host cell and ultimately destroys the host cell; the phage DNA penetrates the cell, destroys the host chromosome, replicates itself within the cell, and then destroys (lyses) the cells as new phages are released

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Virulent Phages

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referring to a virus of microorganism that can be extremely damaging to a host; they are released during the lytic cycle

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Lysogenic Cycle

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The events of a bacterial virus infection that result in the integration of its DNA into the bacterial chromosome; the phage DNA integrates into the host chromosome as a prophage and the phages participating are temperate phages. The host cell survives, and as it undergoes DNA replication and binary fission, the prophage is copied and vertically transferred to daughter cells

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Lysogenic Cycle

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The events of a bacterial virus infection that result in the integration of its DNA into the bacterial chromosome; the phage DNA integrates into the host chromosome as a prophage and the phages participating are temperate phages. The host cell survives, and as it undergoes DNA replication and binary fission, the prophage is copied and vertically transferred to daughter cells

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Prophage

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the viral DNA of a bacterial virus that is inserted into the bacterial DNA and is passed on from one generation to the next during binary fission

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Temperate Phages

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referring to a bacterial virus that enters a bacterial cell and then the viral DNA integrates into the bacterial cell’s chromosome

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Generalized Transduction

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A process by which a bacteriophage carries a bacterial chromosome fragment from one cell to another

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Generalized Transduction

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A process by which a bacteriophage carries a bacterial chromosome fragment from one cell to another

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the host cell's chromosome is digested into small fragments. When the new phage DNA is produced, the DNA normally is packaged into new phage particles

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After having DNA injected into the cell, what happens in the host cell?

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defective cells

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On rare occasions, a random (general) fragment of host cell DNA may accidently be captured in the packaging process and end up in a phage head rather than the phage DNA. They are fully formed and can infect another cell, they are called ___ b/c they carry no phage genes and cannot replicate themselves after infection.

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defective cells

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Following release from the lysed host, ____ attaches to a new (recipient) cell and injects the donor chromosomal DNA into the host cell. Once in the recipient, new genes can pair with a section of the recipient’s DNA and replace the section in a fashion similar to conjugation, the recipient has now been transduced using genes from a donor cell

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Specialized Transduction

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the transfer of a few bacterial genes by a bacterial virus that carries the genes to another bacterial cell; as a result of lysogenic cycle, results in transfer of specific genes

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Specialized Transduction

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diphtheria bacillus toxin Corynebacterium diphtheriae, staphyloccoal enterotoxins in food poisoning, clostridial toxins in some forms of botulism, and streptococcal toxins in scarlet fever, harbor proviral DNA providing genetic code for the toxins. What are these examples of?

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Biotechnology

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the commercial application of genetic engineering using living organisms

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Recombinant DNA molecule

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A DNA molecule containing DNA from two different sources

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Restriction endonuclease

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A type of enzyme that splits open a DNA molecule at a specific restricted point, important in genetic engineering techniques; recognizes and cuts specific short stretches of nucleotides, leaving mortise like staggered ends

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Sticky Ends

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These staggered ends dangling bits of single-stranded DNA extending out from the double-stranded DNA easily attached to complementary ends protruding from another fragment of DNA

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Palindrome

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A series of letters reading the same left to right and right to left

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DNA Ligase

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An enzyme that binds together DNA fragments; seals the recombinant DNA segments, but normally functions during the DNA replication and repair to seal together DNA fragments

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Clones

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A population of genetically identical cells (or plasmids)

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Results from Genetic Engineering
Commercial and Practical Applications

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Diabetes a group of diseases resulting from abnormally high blood glucose levels. One is caused by the inability of the body to produce sufficient levels of insulin to control the blood glucose level. Hummulin was the first synthetic human insulin. What is this a result of and an example of?

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Bioremediation

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The use of microorganisms to degrade toxic wastes and other synthetic products of industrial pollution, like waste materials or oil pills in attempt to return the environment back to its original condition

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Genetic Engineering
Medicine

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microorganisms used to produce a larger quantity of antibiotics and/or produce modified antibiotics to which infectious microbes have yet to show resistance. This results from? and is an example of?

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Interferon

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An antiviral protein produced by body cells on exposure to viruses and which trigger the synthesis of antiviral proteins; a set of three naturally produced antiviral agents produced by the human body, two of which block viral replication

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Vaccines

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_____ are safer as a result of genetic engineering because they can now contain a part of the whole microbial agent, or isolating a gene that will stimulate the immune system to generate protective immunity. This means that a patient is not exposed to the active virus or bacterium that can cause disease

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Genes for herbicide resistance

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These for this have been transplanted from cloned bacterial cells into tobacco plants, demonstrating that these transgenic plants better tolerate the herbicides used for weed control

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Transgenic

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referring to an organism containing a stable gene from another organism

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Genes spliced

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from a pathogenic virus into tomato plant cells and demonstrated that the cells would produce viral proteins at their surface. The viral proteins blocked viral infection, providing resistance for the transgenic tomato plants

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Resistance to insect attack

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This has been introduced into plants using a plasmid carrying a bacterial gene that is toxic to beetle and fly larvae

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practical and commercial application of genetic engineering in agriculture

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Food can be changed so that it tastes better, grows faster, and larger, or has a longer shelf life

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Argobacterium tumefaciens

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In many experiments, DNA often used in a plasmid, which causes a plant tumor called crown gall, which develops when DNA from the bacterial cells inserts itself into the plant cell’s chromosomes, so by removing the tumor inducing (Ti) gene from the plasmid and then splice the desired gene into the plasmid and allow the bacterial cells to infect the plant; this system works with dicots, like tomatos, potato, soybeans, and cotton. What type of microbe is used in these experiments?

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DNA Probes

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A short segment of single stranded DNA used to locate a complementary strand among many other DNA strands; single stranded DNA molecules that recognize and bind to a distinctive and unique nucleotide sequence of a pathogen

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Polymerase Chain Reaction (PCR)

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A technique used to replicate a fragment of DNA many times

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Polymerase Chain Reaction (PCR)

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The DNA probe binds (hybridizes) to its complementary nucleotide sequence from the pathogen, like velcro straps stick together. To make a probe, scientists first identify the segment or gene in the pathogen that will be the target of the probe. Using this segment, they construct the single-stranded DNA probe

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DNA Probes

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What are used for HIV, HPV, and E.coli detection?

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DNA microarray

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A series of DNA segments used to study changes in gene expression

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