Rhyme Case Study

The guiding question relates to our background info because in order to know the effect the different solution had on the cell size we had to know our terminology, which was included in the background information. Section2: For this lab we had to follow certain procedures.

In order to achieve this goal they created variants of each gene. These genes were all removed by a similar process by which the pieces of the gene were cut with different restriction enzymes and purified and cloned in order to give us different plasmids. They were amplified using PCR and these were then integrated into the regions and then grown on media and observed. When there was excision of the gene they were looking to remove these strains were cultivated and given designations. Strain WR 441(hDHFR-2) has the hdrA was deleted, WR445 has a deletion of both genes and WR446 (hDHFR-1) has a deletion of hdrB, WR 447 had hts deleted from it. These strains in turn were grown on three different Medias HY which was the rich media, M which was the minimal media and agar plates. In these experiments they found that WR441 needs nothing else and grew on each media with no other enrichment. However WR446 and WR 445 could grow on minimal medium containing both trimethoprim and thymidine, but only when that medium was supplemented with glycine, methionine, pantothenic acid and hypoxanthine. Another strain WR447 was grown and found that it was auxotrophic for thymidine. However when plasmid pHE4 was added to it could grow on minimal medium containing trimethoprim and thymidine. When the hts was removed the hdrB gene did not have a strong promotor which appeared when the hDHFR-2 didn’t express itself enough to give the organism the trimethoprim resistance it need to get a high copy number. Because of this we can use the hdr gene as an expression point for Hf.volcanii. At the lowest level of expression, only complementation of purine, glycine, pantothenate and methionine autotrophy will occur When the gene has a higher level of expression we can see that there will be prototrophic growth in minimal medium, and when the gene is working at its highest level it will give a resistance to

The motif was compared with its ancestral sequence to test, and further validate, whether it is a good binding motif. The similarities between and ancestral were compared to the ones between motif and random promoter sequences. The results showed that in most cases, a good biding motif was embedded in an ancestral repeat instead of a random promoter sequence; this confirms that the ancestral repeats were more easily able to produce a good binding

As the target gene will be incorporated into the Pichia genome via homologous recombination, each transformant may have varying numbers of the gene incorporated into their genome.

It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region. In E. coli two recombinases are required for recombination at the dif site. Xer C and Xer D behave as site specific type I topoisomerases and relaxes the supercoiled plasmids. Without the homologous recombination stalled or broke replication forks wont reform and wont re-initiate replication. (5) If the dif is not located in the terminus region for proper function and may be replaced by loxP if the Cre recombinase is supplied. To clarify, if the dif/Xer system fail the Cre/lox system can be used in its place otherwise recombination will not occur. Cre recombinase recognizes specific DNA sequences and catalyzes recombination even if the chromosomes are not homologs. When Cre is expressed it binds to loxP sites and forms a dimer. When the dimer binds to another loxP site, it forms a tetramer. The strands are cut by the Cre protein and rejoined by DNA ligase. The orientation of the LoxP sequence matters, because it would determine whether Cre will produce a deletion, inversion, or translocation. Two LoxP sequences that go in the same direction would be recognized and cleaved by Cre, producing a deletion. If the LoxP sites are oriented in opposite directions, Cre would produce an inversion. If the LoxP sites are in different chromosomal loci, Cre

A Biobrick is a specific sequence that is inserted into a plasmid. The Biobrick’s restriction enzymes are standardized and are the same set of restrictions enzymes, so the parts can be easily ligated together. There are 2 conserved elements in promoters -35 and -10 sequence. Bacterial polymerases require a sigma factor to bind to promoter sequence, and variations in these sequences as well as in the spacing between them affect how well the sigma factor binds, leading to different promoter strengths.

(2) Homologous recombination replaces the target gene with the insert gene provided. Therefore, the insert gene must contain flanking regions homologous to the regions surrounding the target gene. There are three main steps to gene targeting: 1. Synthesis of the primer for cloning the target gene, 2. Cloning the target gene from a source by PCR, 3. Introduction of the target gene into the cell. Now the recombination can occur to replace the target gene with the insert

The phenotypic rescue of Grp170a deficient C. elegans will be performed using mouse Grp170 cDNA that will be introduced via vector. Research will be done in order to obtain the adequate plasmid needed in order to perform the experiment. Once the vector is identified, primers will be designed in order to adequately encapsulate restriction enzyme sites on the mouse Grp170 gene. These primers then will be used to preform high fidelity PCR of the desired gene. Once the desired PCR product is obtained, a DNA restriction enzyme digest will be performed. This will be followed by a plasmid restriction enzyme digest; both the digested DNA and plasmid then will be ligated together. The transformation of NEB 5-alpha competent E. coli cells will be done in order to produce plasmid DNA. The plasmid DNA will then be purified and an electrophoretic analysis will be performed to ascertain the presence of DNA in the plasmid.

Through the use of plasmid removal and cloning, bone marrow cell culture, localization microscopy, and analysis of

In conclusion, the poem “A Fit Of Rhyme Against Rhyme “by Ben Johnson is about how poetry should rhyme. A man named Thomas Campain wrote an essay saying that poetry shouldn’t rhyme. Ben Johnsons responds was writing a sarcastically rhyming poem with wording of talking trash to Thomas because he said he wouldn’t know how to write poetry because he never wrote any. Ben stated that poetry should never rhyme, don’t u believe the same thing? don’t you believe poetry should

Genetic transformation has the ability to alter genes, thus leading to the expression of different phenotypes. This technique can be used in a variety of sectors of biotechnology to help advance things such as agricultural production, bioremediation, and medical treatments. In this lab, we attempted to successfully complete genetic transformation on an Escherichia Coli sample, by getting the bacterium to express the green fluorescent protein, which is a protein that comes from a species of jellyfish called Aequorea victoria. The bacterial transformation method we used in this lab is made possible because bacteria take up free DNA from their environments. Bacteria also contain circular pieces of DNA called plasmids. These plasmids allow genes to be transferred more easily, and can lead to sharing of beneficial genes.

Injection of DNA into cells which results in the inheritance of traits contained in the DNA injected is known as genetic transformation (Lorenz et al, 1994). Genetic transformation that occurs naturally has resulted in evolution. The efficiency and rate of exchange of genetic material can be increased if both organisms use the same system of genetic information storage e.g. DNA plasmid (Johnsborg et al, 2007). The bacteria E.Coli which are used in the experiment are sensitive to antibiotic, they do not glow and are easily transformed (Dickson, 2008). Bacteria are single celled which increases the result of an uptake of substance, and E.Coli have circular DNA or better known as plasmids. These plasmids can be replicated and passed on to the next generation. The plasmids added to the colonies will be used as the medium for insertion and expression of the foreign DNA sequence (Cold Spring Harbor Laboratory, No Date). A gene carrying the GFP or Green Fluorescent Protein is used in the experiment. GFP is found naturally in the North American jellyfish, Aequorea Victoria, and is harmless hence leading to its usage as a tool to study gene expression in an organism as they are easily visible (Dickson, 2008). GFP causes an organism to glow under UV light.

Genetic engineering has developed to target specific modifications in the genome of living organisms for a desired outcome. To assist in the process, the CRISPR/Cas9 system has been developed to create insertions and deletions in chosen genes allowing for the alteration and analysis of phenotypic changes resulting from the targeted mutations (Bassett et al., 2013). This is significant for determining the role of various proteins, examining methods to insert new genes into organisms, and for the potential to examine human disease models in organisms. For the purposes of this review article, the CRISPR/Cas9 system will specifically be talked about with regards to genetic modification of the Drosophila fruit fly due to the flies relative short life span and ability to design experiments over multiple generations in short time periods, as well as a comparison of the CRISPR/Cas9 system to alternate methods of genetic engineering.

The tubes were then centrifuged at 1,000 rpm for 3 minutes to remove the large tissue pieces. The supernatant of this tube contained the soluble compounds and proteins that were present in the cytosol of the cells present in the tissue. The supernatant was removed using a micropipette and transferred to a new 1.5 mL-Eppendorf tube (Tube B), and the pellet Tube A was discarded.

Cells were lysed with Laemmli buffer, and each lysate sample was loaded into a polyacrylamide gel and separated by electrophoresis at 30 mA following by transfer onto PVDF membranes (Millipore). Membranes were blocked for 1 h at room temperature using 5 % skim milk in Tris-Buffered Saline with 0.1 % Tween-20 (TBST). WB analysis was