Ligation Analysis Lab

The pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the sugar arabinose as well as the ampicillin resistance gene β-lactamase (bla) (Brown, 2011). Original E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on LB and LB/amp agar plates and all four plates were incubated at 37°C for

Control plasmids lux and pUC18 were introduced into E. Coli through a process of transformation.

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).

The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

Our results show no growth whatsoever in those plates containing Ampicillin; this indicates that we encounter an error during our experiment. The agar plate's outcomes and bioluminescent response done by the bacterium that had the plasmid, it can be presumed due to scientific analysis that Escherichia coli is impervious to ampicillin and the plasmid combines itself with the DNA of Escherichia coli according to other experiments and based on science itself. We can predict that the impact of the bioluminescence in the cells of the microorganisms that is infested unmistakably gives affirmation that the plasmid infuses with Escherichia coli's DNA, guarding the cells that changed from dying, viably creating a gainful situation for the bacterial organisms. Since Escherichia coli is a negative prokaryotic call, it is within the phospholipid bilayer and on top of this is a peptide glycan

Coli bacteria, and mix it with the plasmid. We can select the bacteria that are resistant by introducing ampicillin. Bacteria that are not resistant to ampicillin dies. The screening of the bacteria with the resistance occurs when the bla gene turns on to produce beta lactamase which will kill the antibiotic ampicillin. After this, we can get the plasmid into the bacterial cell through the process of horizontal gene transfer through transformation. This can be done by a cold treatment in an ionic solution, then a heat shock where it increases membrane fluidity to take up the plasmid into the bacteria cell. In order to know if the EPAS1-TD gene product was produced by the bacteria, we have to perform a Western Blot. We do this by extracting the protein and run it through gel electrophoresis to separate the proteins by their size. We then label the proteins with the colored probe based on their size. We can detect the EPAS1-TD gene being produced based on the colored

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

Each one recognizes a unique sequence of 4-8 nucleotides in a DNA molecule and are cut at specific locations within the site. The cutting is done by the use of restriction endonucleases, also called enzymes. Hundreds of restriction enzymes have been identified. “A bacterium carrying such a plasmid is able to live and multiply in the presence of an antibiotic, while cells lacking the plasmid are unable to divide. Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities.” (A Kuspa, WF Loomis) In the following experiment a recombinant plasmid was created. A restriction enzyme was chosen based on the presence or absence of a gene (LacZ). The LacZ gene codes for an enzyme called beta-galactosidase.

A careful comparative sequence analysis revealed that most of the Vibrio species formed a phylogenetically tight group. However, V. vulnificus and V. parahaemolyticus branched off separately from the main cluster. This finding is not in complete agreement with a phylogenetic tree inferred from a comparative analysis of 16S rRNA sequences obtained by reverse transcription of cDNAs from Vibrio species Gauger and Gomez- Chiarri 2002). According to the data of Dorsch et al. (1992), the V. vulnificus is more closely related to V. parahaemolyticus and belongs to the main cluster of Vibrio species. The two species are indistinguishable by the conventional serological methods. This close relationship is confirmed by the high level of 16S rRNA similarity of the two species. The findings of Gauger and Gomez- Chiarri (2002) and of Thompson et al. (2003) could be reinforced in this study as the results obtained in this study reflects that none of the characters of phenotypic and genotypic, clearly discriminate the two species and it can be said that phenotypic character is not sufficient in differentiatiating these species. Bootstrap for some clusters was found to be lower than 30. However, except for three cases, we have found 100% query cover in the BLAST analysis (Table 5). Therefore, it is recommended that the molecular fingerprinting

Today, we are using Biotechnology to by-pass nature 's random fashion of generating variation. We will be genetically engineering a new organism through a process known as transformation (transfer of genetic information between organism’s takes places via the movement of an extracellular piece of DNA). Bacteria are great for experiments for genetic manipulation simply because of how long they take to produce, their simplicity, and how easily recognizable they are. The bacteria that we will be using for this experiment is Escherichia Coli, also known as E. coli. Most of this bacteria genetic information is carried on small pieces of extrachromosomal DNA. The small DNA molecules are known as plasmids. Plasmids carry genes that allow bacteria to neutralize antibiotics that would typically kill them only one molecule in 10,000 can successfully transform a bacterium in the most favorable conditions in the laboratory, so for it happen in nature by itself is incredible. It is a true test of survival of

The genomic DNA sample concentration of 28.5 ng/μL and its A260/A280 ratio of 1.85 indicates that the DNA was relatively pure since a 260/280 ratio of ~1.8 is generally accepted as pure for DNA (Cox, Doudna & O’Donnell, 2015). The ratio of white colonies to blue colonies observed on the blue-white screen plate was 31:37, which shows that there was a greater number of E. coli DH5-α that did not contain the plasmid with the foreign gene insert. Therefore, out of all of the E. coli DH5-α cells that were plated and were transformed successfully, there were more cells containing the plasmids without the foreign insert than there were plasmids with the foreign insert.

In this experiment, LacZ DNA segment was isolated by PCR and ligated to pET32a vector plasmid for further use in future experiments. LacZ gene has been used in wide range of experiments in the Biochemistry field. The gene produces the -galactosidase enzyme that is malleable and easy to manipulate, making it a great subject for experiments to understand the molecular genetics of prokaryotes (Serebriiskii and Rose et al.). The LacZ gene was isolated from E.coli bacteria by PCR because it is an efficient and affordable method of amplifying a specific gene. The PCR product was ligated to a vector plasmid, pET32a, so it can be inserted to a bacteria cell to be expressed and analyzed. Restriction digestion with the same enzyme was done to the LacZ gene and vector plasmid prior to ligation. This step ensures that the two will ligate at a specific position.

The plasmid DNA will be used integrated into the genome of the E. coli bacteria. To generate the effects of the plasmids and transformation, the

Our device consists primarily of two genes that encode enzymes PchA and PchB following an IPTG inducible promoter. The coding region is preceded by a strong RBS binding site. The gene for PchB is preceded by a gene which produces a His tag, causing the protein to be fused to the tag and allowing for experimental observations of the enzyme. The gene for PchA is followed by a gene encoding for a flag tag, causing the protein to be fused to the tag and allowing for experimental observation. The coding region is followed by a terminator. The plasmid also includes an origin of replication and genes for Kanamycin resistance.