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Testing for Antibiotic Resistance

INTRODUCTION:
Plasmids are circular, self-replicating, double -stranded extra chromosomal DNA molecules. E. coli plasmids can be tailored for its extensive use as cloning vectors in Recombinant DNA technology because of its genetic simplicity. (Sandy. B Primrose, Richard M Twyman, Robert W Old 2001) These plasmids usually have a high copy number, low molecular weight, ability to render selectable phenotypic traits and a single site can incorporate a number of restriction endonucleases. In addition to these essential constituents required for cloning, plasmids also contain a replication origin, an antibiotic resistance gene and a region in which the exogenous DNA can be inserted. (Lodish, 2008)
AIM AND OBJECTIVES:
To start with the subcloning of PstI fragment from the pMB into pUC 19, the plasmids pMB and pUC 19 are digested with PstI restriction enzyme. The restriction fragment is then isolated from the pMB and the vector pUC 19 is treated with shrimp alkaline phosphatase to remove the 5’ends of the plasmid. The DNA is then purified using selective column binding followed by the ligation of the insert fragment and vector to produce the recombinant pUC19. In the meantime, competent E. coli XL1 blue (tetracycliner) competent cells are prepared and are tested for efficiency. These cells are then transformed with the DNA molecules and are eventually screened (blue/white X-gal screening) for recombinant bacterial colonies on agar plates (Ampicillin and IPTG induced). Finally, the recombinant bacterial colonies are picked to conduct a small scale plasmid preparation which successfully terminates the desired subcloning. The insert fragments can now be released by digesting the recombinant plasmid with PstI and analysed by electrophoresis.
Molecular basis of a-complementation and blue/white selection:
The pUC plasmids are the most widely used cloning vectors with an easily selectable ampicillin resistance and the ease of insertional inactivation. It encodes for the N-terminal region of the LacZ gene (a-peptide) that complements the C-terminal of Lac Z ?-peptide turning the initially inactive one to active. This turns the colonies blue on X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). Here the host chromosome codes for the remaining ?-peptides that produces the enzyme ß-galactosidase after tetramerization. This phenomenon is called a-complementation. (Nelson et al. 2008) When a foreign DNA is inserted at the Lac Za gene, the production of the ß-galactosidase enzyme gets inhibited (no production of a-peptide)and hence the production of white colonies. This is mainly due to the loss of the recombinant cells’ ability to hydrolyse the markers. The Lac region is usually inducible and is turned on by the use of IPTG (isopropyl-ß-D-galactosidase). (Griffiths 2007)
Molecular basis of Ampicillin resistance:
Ampicillin is atype of ß-lactam antibiotic. A given bacterium is usually made antibiotic resistance by transduction, transformation, conjugation or fusion with the methods having its own limitaions. The objective is to genetically transfer a ß-lactamase encoding gene called the bla gene via plasmid transfer, if not present inherently in the bacterial chromosome, which on hydrolysis confers the resistance to the bacterium. (Atlas 1988) This is also mediated by transposition wherein the transposon (a DNA unit), can move within the same structure in a bacterial cell carrying the gene specific for an antibiotic resistance from one self-replicating DNA to another. In the case of ampicillin, a Tn3 unit confers resistance to it due to the presence of the bla gene. The structure usually moves as a whole, inducing changes in the genetic composition of the bacterium, offering resistance to ampicillin. This spread of ampicillin resistance is generally monitored at any of these three stages transposition step, an extrachromosomal unit (if the gene is present in the chromosome, celll-cell transfer) and at the point of infection. The ampicillin also competes for the Transpeptidase enzyme which aids bacterial cell wall synthesis and eventually leads to lysis. This inhibition is done by the target modification of the outer bacterial cell wall and the penetrability through the cell wall is varied to accommodate other essential metabolites too. (Gale 1981)
RESULTS:
The strains of pMA and pMB with three others (DH5a, XL1 blue, pUC 19) are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. pMA is found to be both ampicillin and tetracycline resistant whereas pMB is found to be tetracycline and kanamycin resistant. The PstI fragment subcloned from pMB contains Kanamycin as an antibiotic resistance, this cannot be tetracycline because when this antibiotic meets the cell wall of a sensitive bacteria (pUC19-amp resistant) it has an initial free inflow and outflow which is later followed by a block of the outflow leading to accumulation of the tetracycline within the cell. To optimize on its flow into the cell, there must be an alteration in the peripheral membrane proteins which require suitable induction. (Gale 1981)
Competent E. coli XL1-blue cells were prepared and stored at -800c after the addition of glycerol. The transformation efficiency of the competent cells were calculated.
50µl of 1ng pUC19 DNA: 8. 2*106
50µl of 5ng pUC19 DNA: 1. 94*106
200µl of 1ng pUC19 DNA: 7. 25*106
200µl of 5ng pUC19 DNA: 1. 82*106
Gel purification of both the insert fragment and the PstI-cut pUC19 vector DNA were carried out and ligated. The ligated samples were transformed with the competent cells to grow recombinant cells on LB agar plates containing ampicillin, IPTG and X-gal. (Huff et al. 1990)
Small scale plasmid preparation of the potential subclones was set up and the restriction analysis of plasmid DNA was carried out by releasing the insert fragment from the vector by digesting the recombinant plasmid with PstI.
DISCUSSION:
Hence the PstI fragment from the pMB got successfully subcloned into the pUC19 which is shown by the similar sizes of the insert into the vector DNA molecule during the small scale preparation of the plasmid. Here a Positive selection could have been employed for recovering only bacterial clones with recombinant pUC19 containing the PstI fragment from pMB. This allows only the recombinant clones and masks the growth of the wild-type organism. Otherwise, the use of Selected and Unselected markers can provide similar results with the selected marker to spot the recipient vector DNA that has undergone recombination and also look for other markers they can mutate with and the other markers can be now termed unselected markers. (Snyder

Methods for DNA Extraction Comparison

Lisa Lyons
Abstract:
In this experiment, I extracted DNA from the cells of Green Split Peas and Chicken Livers. I used different variables for each. First, I did the experiment with all materials being cold. The second time I did the experiment with materials at room temperature. My objective was to see which method would extract more DNA. The results were that the materials being colder extracted more DNA than the room temperature materials. In either case, I was able to extract more from the Green Split Peas than the Chicken Livers both times.
Introduction:
DNA is short for deoxyribonucleic acid. Nucleic acid, which is the genetic material determining the makeup of all living cells and many viruses. It consists of two long strands of nucleotides linked together in a structure resembling a ladder twisted into a spiral. The rungs of the ladder are made up of nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). DNA can replicate itself. DNA also serves as a template for synthesis of RNA in the presence of RNA polymerase. (APA, Dictionary. com)
DNA is located in the chromosomes in of the human body. It is like blueprints or instructions for hair color, eye color, height, pretty much everything. DNA can be used to identify criminals with unbelievable exactness when genetic evidence exists. Similarly, DNA can be used to clear suspects and clear persons wrongly accused or convicted of crimes. In all, DNA is ever-increasingly crucial to ensuring precision and fairness in the criminal justice system. (The United States Department of Justice) DNA is used in a few ways to solve crimes. Comparing a suspects DNA to DNA found at the scene, or it can be put in the database to try to find the offender’s match. DNA can also be used to identify victims.
Materials and Methods:
Chicken Livers
Green Split Peas
91% Rubbing Alcohol
Meat Tenderizer
Salt
Blender
4 small glass Pyrex bowels
Small strainer
Wooden Chopstick
Measuring cup
Liquid soap

Put in a blender:
1/2 cup of split peas (with the chicken livers I used 3 medium sized livers. )
1/8 teaspoon salt
1 cup cold water
Blend on high for 15 seconds
Pour through a strainer into measuring cup.
Add 2 tablespoons liquid soap and swirl to mix.
Let the mixture sit for 5-10 minutes.
Pour the mixture into glass bowls, each about 1/3 full.
Add a pinch of meat tenderizer and swirl very carefully
Tilt bowls and pour in rubbing alcohol. (Until it forms a layer on top of mixture)
Results:
The first experiment I used very cold materials, with the peas and with the chicken livers. After blending the peas, I got a very light green colored water solution. After straining, there was a lot of shell like material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. Almost immediately, I was able to see the white colored stringy material in the peas. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. I was able to transfer the DNA to another Pyrex bowl with alcohol in it to get a better look.

After blending the livers, I got a very reddish, beige colored water solution. After straining, there was liver material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. Almost immediately, I was able to see the white colored stringy material in the livers. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top I was able to transfer the DNA to another Pyrex bowl with alcohol in it to get a better look.
The second experiment I used almost room temperature materials with the peas and with the chicken livers. After blending the peas, I got a very light green colored water solution. After straining, there was a lot of shell like material at the bottom of the blender. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. This time there was hardly any of the white stringy material I found in the first experiments, and barely anything to transfer.

After blending the livers, I again got a very reddish, beige colored water solution. After straining, there was a lot of material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. This time there was hardly any of the white stringy material I found in the first experiments, and yet again, barely anything to transfer.
Discussion:
My results were surprising. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top.
It really made a difference in the outcome just by using colder materials. Using ice-cold water and ice-cold alcohol will increase the yield of DNA. “Low temperatures protect the DNA by slowing down the activity of enzymes that could break it apart. The cold alcohol helps the DNA precipitate (solidify and appear) more quickly. Why would a cell contain enzymes that destroy DNA? These enzymes are present in the cell cytoplasm (not the nucleus) to destroy the DNA of viruses that may enter our cells and make us sick. A cell’s DNA is usually protected from such enzymes (called DNases) by the nuclear membrane, but adding detergent destroys that membrane. ” (gslc. genetics. utah. edu)
Out of the two, the warmer water experiments eliminated the most debris, but were not very successful at extracting as much DNA material.
References:
American Psychological Association: dna. (n. d. ). Dictionary. com Unabridged. Retrieved April 12, 2015, from Dictionary. com website:http://dictionary. reference. com/browse/dna
Explorable : https://explorable. com/who-discovered-dna
Learn. Genetics, The Genetic Science Learning Center, Retrieved from: http://learn. genetics. utah. edu/content/labs/extraction/howto/faq/

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