The plate labeled LB/amp/ara: +pGlo had several surviving colonies like the other +pGlo plate, except this time the colonies were a green color and the glowed under a UV light. The glow would be a result of adding the sugar arabinose, which seems to be acting as an inducer in the operon of the bacterium. It can be considered an inducer because in the presence of the arabinose, the colonies glowed, but without it the colonies did not experience any change, leading to the belief that the gene that leads to the green fluorescence is normally turned off. To further the research on the effect of the arabinose on the E. coli, a sample of the bacteria in the LB/amp: +pGlo and placed it in a plate with arabinose lines across the bottom.
Transformation in bacteria usually takes place when a bacterial cell accepts strange DNA and integrates to its own DNA. The transformation normally takes place within plasmids, which are tiny circular DNA molecules that have been separate from its own chromosome. The copies of the same plasmid range from 10 to 200 copies within a cell. These copies of plasmids may multiply when the chromosome replicate or multiply independently. One plasmid has a range of 1,000 to 200,000 base pairs.
The plasmid and the gene are both cut using restriction enzymes. To incorporate the gene into the plasmid, both the plasmid and gen stuck together by a bacterial enzyme called ligase. The plasmid with the foreign gene can then be inserted into the bacteria One specific plasmid is pUB110 which is circular whose host is Bacillus subtilis. It has a plasmid size 2.3 kpb and copy number between 20 - 50. We can selectively grow bacteria that contain this plasmid by using selective and differential media.
Reactions were performed in a 96-well DNA thermo cycler (Eppendorf Mastercycler, Germany) using the following reaction mixture: • 2.0 μl of genomic DNA (10 ng/μl) • 1.5 μl of 10x PCR buffer (NH4 Reaction buffer, Bioline) • 1.5 μl of dNTPs (0.2 mM) • 0.9 μl of 50 mM MgCl2 (Bioline) • 0.9 μl of each forward and reverse primer (2 mM) • 0.15 μl (5 u/μl) of Taq DNA (Bioline, Australia) • 7.15 μl of
Results Figure 1 This image to the left represents the gel in the gel electrophoresis chamber before being run. As seen here, the DNA samples of the WD, WU, MD, and MU were all underfilled. In other words, there was not enough sample loaded. This was a potential error that could result in no bands after electrophoresis.
15 mL of Solution A and B were mixed together to form solution F. Eight cuvettes were labeled distinctly as 1a, 2a, 3a, 4a, 1b, 2b, 3b, 4b, where “a” cuvettes were used for the concentration experiment and “b” cuvettes were used for the temperature experiment. Cuvette 1, the blank tube was prepared and the spectrophotometer was set to 405 nm. The enzyme was added, upon being ready to start the experiment, to tube 1 which then became tube “1a.” 3 mL of solution F was added to each cuvette, both “a” and “b.” The “b” cuvettes were then placed in their specific temperatures, 1b in the fridge, 2b in room temperature, 3b in a 32 degrees Celsius water bath and 4b in a 60 degree water bath. The temperature was recorded using a thermometer that was placed in the surroundings of the tube.
Coomassie Blue Staining measure total protein and immunoblotting measure a specific protein of DnaK. In the Coomassie Blue Staining at 75 kD, exposed for 1 minute, 3 minutes, and 5 minutes appear the induction of heat shock protein. Proteins at 75 kD, exposed to heat shock treatment for 10 minutes and 15 minutes appear the staining of the Coomassie (see Figure 1). Moreover, the polypeptide at 75 kD for 3 minutes changes from the control to the sample (Figure 1). From our analyses of using immunoblotting short duration will not lead the antibody to bind to a specific protein of interest.
The lab, Ester Synthesis, main purpose was to illustrate if chemists can create different smells, from mixtures, in the laboratory. We began by creating a hypothesis from scratch, not knowing anything about what we’re working with, and ended up with a hypothesis which stated the mixtures present for this lab, Acetic acid, Butyric acid for our Carboxylic Group (Putrid smell), and our alcohol listing was isopentanol, butanol, and ethanol. Before we even began the lab, my group and I were already aware that to be a putrid smell, you must have a -ic acid ending to your molecular name or you must have two oxygen atoms present in your structural formula, however, the molecular formula was not needed, so we put that idea to aside. Below you can see
Describe the general structure and shape of an enzyme. In particular, the role of the amino acid R groups in stabilising the shape should be covered. (P4) Enzymes are important catalysts for biochemical reactions. Enzymes can speed up the biochemical reactions by providing another reaction pathway of lower activation energy.
The helicase enzyme initiates the DNA process by unwinding the double helix. If it was missing, the DNA would not be able to replicate as the helix structure would not open. The next step of DNA Replication is the binding of RNA primase in the the initiation point of the 3'-5' parent chain. RNA primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases and also provide a starting point for DNA polymerases to extend from. Without it, the DNA nucleotides would not have something to bind to and to start the DNA synthesis.
Mechanistically, DNA replication in eukaryotic organisms is similar in prokaryotic organisms. Before I examine their differences, I will first describe the general steps to replicate the DNA of E. coli. Scientists have gained much insight into DNA replication by studying this particular bacterium. Unlike eukaryotes with linear chromosomes, E. coli’s chromosome is circular.
Agarose gel electrophoresis is an easy and common technique of separating and analyzing DNA. The main objective of this lab is to find the sire of the offspring using gel electrophoresis. Gel electrophoresis is used in laboratories to isolate charged molecules like DNA, RNA, and particular proteins according to their specific size. The charged molecules travel through the gel when an electric current is spread across it. The electric current is applied across the gel so that the ends of the gel have a positive charge and the other end has a negative charge.
A practical step-by-step on how to transcribe and translate DNA sequence DNA transcription and translation are common terms in DNA replication. Therefore, for one to understand and master how to transcribe and translate a particular DNA sequence, one needs to know the meaning of DNA replication, DNA transcription, and DNA translation. DNA replication is defined as the synthesis of daughter DNA from the parental DNA. DNA transcription is the process of synthesizing RNA using the DNA template. DNA translation is the process of synthesizing proteins using the messenger RNA (mRNA) as the template.
This isotope of nitrogen contains one more neutron within its nucleus causing for it to have a higher atomic mass that 14N. Due to the fact that nitrogen atoms construct part of the purine and pyrimidine bases within DNA, the DNA of E. coli was marked using 15N through growing the bacteria within 15N ammonia salts as the sole source of nitrogen. This then allowed for the parental DNA to be labelled, therefore the next step was the marking of the daughter strands. This was accomplished through the newly marking the newly synthesized DNA strands containing 14N through placing the replicating bacteria within 14N ammonia salts. After a period of time separate samples were taken from the culture of bacteria (collecting DNA with just the heavy isotope, DNA with just the light isotope, and DNA containing both) was mixed with cesium chloride solution CsCL2.