Genetics lab final
What are three mechanisms by which bacteria can exchange genetic information?
1)Transformation 2) transduction 3) conjugation 1) Transformation is the uptake of naked DNA from the environment, rare but normally occurring events competent cells/state of competency-->describes cells that have the ability to undergo transformation We can artificially make membrane of bacteria more porus to increase there competency, two general methods: CaCl2 followed by heat shock, or electroporation (high voltage shoots DNA into cells) 2)Transduction is the transfer of DNA between bacteria by an infectious route, mediated by bacteriophage 3) Conjugation is the unidirectional exchange of DNA that requires cell-cell contact, plasmid mediated event, F factor Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.
What is a cloning vector? What essential features have been engineered into cloning vectors to make the processes of cloning more efficient?
A vector is a carrier for recombinant DNA. A vector is a DNA molecule that is capable of accepting a foreign DNA fragment and of replication itself, along with the inserted fragment in some kind of host cell. Common vectors include plasmids and bacteriophage. Selectable markers like antibiotic resistance have been inserted into vectors to make the process of cloning more efficient. Also the insertion of a polylinker region can serve to aid in visual identification of successful recombinants.
Compare and contrast conjugation and transformation.
Both are mechanisms by which bacteria exchange genetic information. Conjugation is the unidirectional exchange of DNA that requires cell-cell contact, plasmid mediated event, requiring an F factor, while transformation is the uptake of naked DNA from the environment, a rare but normally occurring event.
Successful transformation is a rare event. How does a scientist increase her odds of detecting a successful transformant if the overall efficiency is at best 1 in 1000 bacteria?
By incorporation of a selectable marker gene into a plasmid. A selectable marker is usually a gene that codes for antibiotic resistance in bacteria (Kovach, 2015). The marker selects for rare transformants when the bacteria are grown on an antibiotic containing media. Only cells that have been transformed will have the antibiotic resistance and therefore will grow.
What is catabolite repression? How does it work? Is it a form of positive or negative regulation of gene expression?
Catabolite repression occurs when glucose is present and involves activator proteins. In the presence of glucose, arabinose cannot be metabolized. This is due to glucose inhibiting the synthesis of cyclic adenosine monophosphate or cAMP. Without cAMP, the catabolite activator protein (CAP) cannot bind to its target sequence. Without the cAMP-CAP complex, RNA polymerase cannot bind effectively to PBAD even in the presence of arabinose bonded with araC (Kovach, 2015). When glucose levels are low, cAMP levels are high. When the concentration of glucose is high, the cAMP levels are low because glucose is being metabolized, not arabinose. Glucose must be completely metabolized by the bacteria in order for arabinose to be metabolized. Once glucose is completely metabolized, the bacterium switches its metabolic cycle and activates the genes needed to metabolize arabinose. Catabolite repression is a form of positive regulation of gene expression. In the case of positive control, the genes are expressed only when an active regulator protein, e.g. an activator, is present.
What is meant by compatible cohesive ends? Why would a molecular cloner be interested in sets of RE's that generate compatible ends?
Compatible cohesive ends are "compatible" sticky ends meaning that the DNA contains a single stranded overhang that can anneal with another molecule through complementary base pairing. A molecular cloner would be interested in sets of RE's that generate compatible ends because he could join the ends (due to complementary base pairing) to form a recombinant DNA molecule, such as a vector, that could be used for cloning.
What is GFP and why was its discovery worth the distinction of the recent nobel prize in chemistry?
GFP is coded from an isolated gene sequence from Aequoria Victoria, a bioluminescent jellyfish. The GFP isolated gene sequence becomes the target gene on the pGlo plasmid. The GFP has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread. The Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins.
Compare/contrast selection vs. screening. How are they used in cloning?
In a selection for a phenotype , you search for those organisms that grow under a specific set of conditions; normal "wild type" organism do not grow under these conditions, so only the mutants will appear. In a screening you look to identify those organisms that display the phenotype you desire. In cloning selection was based off incorporation of a selectable marker gene into a plasmid. The marker selects for rare transformants when the bacteria are grown on an antibiotic containing media. Only cells that have been transformed will have the antibiotic resistance and therefore will grow. In cloning screening was used to provide a means of visual identification. Colonies were either blue or white based off if the fragment and the vector were ligated together, creating a functional recombinant vector and disrupting the polylinker enzyme production.
What are RE's and how are they utilized in cloning?
Restriction enzymes are bacterial enzymes that originated from bacteria. They can identify and act on (bind to and cut) recognition sequences in dsDNA. The natural function of restriction enzymes in bacteria is to act as part of the bacteria primitive immune system. When a bacteria cell is attacked by a bacteriophage, the bacterium uses its restriction enzymes to destroy the invading DNA of the bacteriophage. Thus restriction endonucleases are enzymes that restrict the replication of molecules lacking a host-specified covalent modification of DNA.
What is DNA ligase and how is it used in cloning?
To use recombinant DNA technology to "clone" a DNA fragment, it is necessary to ligate the fragment into a vector. DNA ligase acts as molecular glue in the sense that it catalyzes the formation of a phosphodiester linkage in order to join two adjacent DNA fragment. In terms of cloning, it cements the formation of a recombinant DNA molecule or a vector. DNA ligase can catalyze the formation of a phosphodiester linkage between two compatible fragments between the 3' hydroxyl group of one fragments end nucleotide and the 5' phosphate group of the other fragments end nucleotide.
How is transformation efficiency calculated?
Transformation efficiency represents the total number of bacterial cells that express the the target gene. Transformation efficiency=Average number of colonies on the agar plate÷amount of DNA spread on the agar plate (in ug)
A bacterial cell that is capable of transformation is said to be what?
competent cells/state of competency-->describes cells that have the ability to undergo transformation
Describe the process of molecular cloning used for creating recombinant DNA molecules.
dsDNA is digested with restriction endonucleases that cut it into fragments containing YFG while also creating sticky ends on the ends of the fragments. The fragments (now linear) are then isolated by size. A "vector" is then cut with the same restriction endonuclease or endonucleases that create compatible cohesive sticky ends and then through complementary base pairing the fragment(s) (isolated fragment containing YFG) and the vector join together. The two are glued together by ligase and then the recombinant vector is function and can be inserted into an organism to be replicated.
Describe the negative/inducbile system of controlled gene expression of the arabinose operon. What is the inducer? What factor is the repressor and how does it work?
negative control, the genes in the operon are not expressed On pGlo, the promoter sequence controlling GFP expression usually regulates the transcription of a group of genes in E. coli called the arabinose or ara operon. An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. The ara operon encodes proteins needed to metabolize the sugar L-arabinose. The operon is comprised of three structural genes that are regulated by the araC gene product (a repressor protein). The araC protein regulates the arabinose operon in a negative-inducible fashion, meaning that it is always repressing the operon (Kovach, 2015). Arabinose plays a big part in gene regulation. The arabinose operon is constructed of genes that help to produce enzymes that break down arabinose into simpler forms so the bacteria can metabolize the sugar, producing energy. The araC gene codes for a regulatory protein that binds to the operators and controls whether or not RNA polymerase can carry out transcription of the operon, and in the case of this experiment, the inserted GFP gene that downstream of the operon. The araC protein represses the operator region by binding as a dimer to the Oxygen and I sites causing a physical looping of DNA containing the PBAD promoter (Kovach, 2015)(figure 3). When arabinose is introduced to the cell, it binds to the araC regulatory protein (induced state). The binding causes the arrangement or morphology of the arabinose operon to change (figure 4). The change in arrangement prevents the araC repressor protein from binding to the I-site and oxygen. RNA polymerase is then able to transcribe the operon genes along with the GFP genes (figure 5, expression of green fluorescent protein). When arabinose is present, the operon genes and anything downstream from them (GFP gene) are transcribing (figure 4). This is known as the ON state of the gene. When arabinose is absent, the gene is turned off due to the folding associated with the araC protein repressor (figure 3).
What two methods of transformation did we present in the laboratory? Briefly describe these methods. Which method is more efficient ?
two general methods of artificially induced competency: CaCl2 followed by heat shock, and electroporation (high voltage shoots DNA into cells) Heat Shock Heat shock consists of taking cells and placing them in a bath of ice cold CaCl2. The vessel containing the solution is then submerged in 42 degree Celsius water for sixty to ninety seconds. The sudden increase in the temperature, hence "heat shock", creates a pressure difference between the outside and the inside of the cell. The pressure difference creates the formation of pores, through which supercoiled plasmid DNA can enter. After returning the cells to a more stable temperature, the cell wall stabilizes. This method is harsh on the cell due to the abrupt temperature change (JoVE Science education database, 2016). Electroporation Electroporation consists of mixing cells with target DNA and then exposing the mixture to pulses of high voltage electricity. The pulses range from 1500 to 2500 volts for a microsecond.
