Chapter 12- Modern applications of microbial genetics Flashcards
Biotechnology
The science of using living systems to benefit humankind. It is associated with genetic engineering
Genetic engineering
The direct alteration of an organism’s genetics to achieve desirable traits. It involves recombinant DNA technology
Recombinant DNA technology
The process by which a DNA sequence is manipulated in vitro, creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism
Transgenic
An organism that has recombinant DNA that was introduced from a different species. One example is the bacterial strain that produces human insulin
How is insulin produced?
The insulin gene from humans is inserted into a bacterial plasmid. The recombinant DNA plasmid was then inserted into bacteria, making it transgenic. The bacteria can then produce and secrete human insulin
Molecular cloning
A set of methods used to construct recombinant DNA and incorporate it into a host organism
Restriction endonucleases
Restriction enzymes- bacterial enzymes that are produced as a defense mechanism to cut and destroy foreign cytoplasmic DNA that typically occurs due to bacteriophage infection. In biotechnology, they are used to cut DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules
Recognition site
A specific palindromic DNA sequence that is between 4 and 6 base pairs. A restriction enzyme recognizes the DNA palindrome and cuts each DNA backbone at identical positions in the palindrome
Sticky/blunt ends
Some restriction enzymes cut to produce molecules that have complementary overhangs called sticky ends. Others cut without producing overhangs, making blunt ends
Hybridization
Refers to the joining together of two complementary single strands of DNA. This can be done more easily in molecules with sticky ends, because the sticky ends can anneal
Anneal
Form hydrogen bonds between complementary bases (at their sticky ends)
Ligation
When DNA ligase rejoins the two sugar phosphate backbones of DNA through covalent bonding, making the molecule a continuous double strand
Vectors
DNA molecules that carry DNA fragments from one organism to another. Plasmids are used as vectors and can be genetically engineered to have specific properties
Polylinker site
A short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Multiple restriction enzyme recognition sites in the polylinker site makes the plasmid vector versatile, and it can be used for multiple different cloning experiments
Reporter gene
A gene sequence that is artificially engineered into the plasmid. It encodes a protein that allows for visualization of DNA insertion. Researchers use it to determine which cells are host cells with recombinant plasmids and which contain the non-recombinant plasmid vector. The polylinker site is often found within a reporter gene
Transformation
A process in which bacteria take up free DNA from their surroundings. In nature, free DNA comes from other lysed bacterial cells
Molecular cloning using transformation
In the laboratory, free DNA is the form of recombinant plasmids is introduced to the cell’s surrounding. Some bacteria are naturally competent and are able to take up foreign DNA. Bacteria can also be made artificially competent by increasing the permeability of the cell membrane. This is done through chemical treatments or through exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. After transformation protocol, bacterial cells are put into a medium containing antibiotics to inhibit the growth of host cells that didn’t get the antibiotic resistance plasmid
Molecular cloning using conjugation
F plasmids are transferred between bacterial cells using conjugation. Recombinant DNA is transferred through conjugation when bacterial cells containing a recombinant F plasmid are mixed with bacterial cells that lack the plasmid. The F pilus is encoded by the plasmid and forms a bridge between the cells. The F plasmid containing cell replicates its plasmid and transfers a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate the transfer of the recombinant F plasmid to an additional cell.
Molecular cloning using transduction
DNA fragments of interest are engineered into phagemids. Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Depending on the type of phage, the recombinant DNA can be integrated into the host genome or it can exist as a plasmid in the host’s cytoplasm
Phagemids
Plasmids that have phage sequences that allow them to be packaged into bacteriophages
Genomic library
A complete copy of an organism’s genome contained as recombinant DNA plasmids, engineered into unique clones of bacteria. Researchers can create large quantities of each fragment by growing the bacterial host for that fragment
How are genomic libraries created?
By ligating individual restriction enzyme digested genomic fragments into plasmid vectors that have been cut with the same restriction enzyme. Each bacterial cell has a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study.
Complementary DNA (cDNA)
All cells have the same DNA, but only certain genes are expressed in certain tissues, and only the genes that are expressing a trait are synthesizing relevant mRNA. mRNA can’t be cloned directly, so in the laboratory, mRNA is used as a template by the reverse transcriptase enzyme to make cDNA. A cell’s full complement of mRNA can be reverse transcribed into cDNA molecules, which can be used as a template for DNA polymerase to make double stranded DNA copies, and can be used to make a cDNA library.
What is the benefit of a cDNA library?
The benefit of a cDNA library is that it contains DNA from only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library.
Transfection
The introduction of recombinant DNA molecules into eukaryotic hosts, mainly plants and animals
Why is transfection of eukaryotic hosts more challenging?
Eukaryotes aren’t competent to take up foreign DNA and they can’t maintain plasmids like bacteria are able to. Plant cells are also more difficult to transfect than animal cells because they have thick cell walls
Electroporation
An electric pulse of a cell culture induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a brief positive charge on one side of the cell and a negative charge on the opposite side. The charge difference draws negatively charged DNA molecules into the cell. This is a method of transfection in eukaryotic cells
Microinjection
Another method of transfection. Eukaryotic cells are larger than prokaryotic cells, so DNA fragments can sometimes be directly injected into the cytoplasm using a micropipette
Gene guns
A method of transfecting plant cells. Plant cells are treated with enzymes to remove their cell walls. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts. Recipient protoplast cells can then recover and be used to generate new transgenic plants
Shuttle vectors
Plasmids that can move between bacterial and eukaryotic cells