The purpose of the lab was to transform E.coli using the plasmid pRFP to promote the expression of antibiotic resistance as well as expression of the red fluorescent protein (RFP). The hypothesis was that if the transformation was successful, then the bacteria would express RFP because the arabinose would activate the plasmid’s red fluorescent protein, and show growth because pRFP allows E.coli to grow even in the presence of an antibiotic. The plasmid was combined with a sample of E.coli through the process of transformation. Following transformation, the protein was isolated using hydrophobic interaction chromatography (HIC) and the size was determined using SDS-PAGE. The results showed that the E.coli transformed by the pRFP would thrive and express the RFP in the proper environment. Through the use of HIC and SDS-PAGE the size of the plasmid was found to be ~27 kDa. Introduction
The purpose of this lab series was to transform E.coli using pRFP and examine the
…show more content…
A lysozyme was used to break down walls of the bacteria to allow the RFP to be released. It was hypothesized that after the solution was put through the HIC column, the RFP would be isolated from the bacteria. The HIC matrix binds to the hydrophobic molecules of the protein in high salt conditions. Red fluorescent protein is hydrophobic while bacterial proteins are not. The bacterial proteins will pass through the column while RFP will stay in the column. The first solution that was added, the supernatant, had a high salt content which made the protein bind to the matrix in the column. The second solution, the wash buffer had a lower salt concentration which helped to keep the protein in the the matrix but allowed it to move down and the solution dripped out. The last solution, TE buffer contained no salt which finally led the protein to drip out into the last collection
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
ABSTRACT: Water samples from local ponds and lakes and snow runoff were collected and tested for coliform as well as Escherichia coli. Humans as well as animals come into contact with these areas, some are used for recreational activities such as swimming and some are a source of drinking water for both animals and humans The main goal of this experiment was to see which lakes, snow run off and ponds tested positive for coliform or Escherichia coli and to come up with some reasoning as to why. It was found that the more remote pond with less contact contained the most Escherichia coli. However, another lake that many swim in and use as their drinking water indeed tested positive for a small amount of Escherichia coli. The two samples from the snow showed negative results for both coliform and Escherichia coli and the two more public ponds that aren’t as commonly used as a source of human drinking water but animal drinking water tested in the higher range for coliforms but in the little to no Escherichia coli range. It was concluded that the remote pond should be avoided as it’s not a safe source of drinking water for humans or animals. Other than that, the the other ponds are likely to be safe from Escherichia coli, but coliforms are a risk factor.
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
Ligating the EGFP cDNA into a pET41a (+) plasmid in order to create recombinant expression plasmids and run these ligations through gel electrophoresis to visualize the DNA and check the success of the ligations. Five ligation reactions were generated, two actual ligations and three controls, with a total final volume of 20uL each. NcoI and NotI are restriction endonucleases whose purpose are to reduce non-recombinant plasmids from forming and to prevent undesired rearrangements during ligation.
Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
tube 1 – 5.8 ml pH5 buffer. 1 ml 0.1M succinate. .2 ml enzyme tube 2. -5.8 ml pH 7.3 buffer
This experiment was very successful as a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. Within the gel electrophoresis, it is necessary to run a 1kB ladder in the first well. This ladder contains numerous known lengths of base pairs, and is run next to and unknown product in order to approximate the sizes of unknown fragments simply by comparing the unknown fragments to the coinciding fragments of the known ladder. This ladder gives us the ability to precisely and accurately draw conclusions about the results derived from the gel electrophoresis as it serves as an essential reference point. Because of the known fragments in the ladder, we were able to create a standard curve. Within the standard curve, the distance the fragments traveled was plotted against the length of the known base pairs within the ladder. Once the points were plotted, a line of best fit was constructed and an equation of the line was electronically derived. By plugging in the measured distance of how far the fragments traveled, shown by “x”, into the equation for the line of best fit, the lengths of the base pairs created by the restriction enzymes was able to be calculated.
Eukaryotic microorganisms can be defined as organisms whose cells contain a nucleus and other organelles enclosed in membranes. It is said that all multicellular organisms are eukaryotes which mostly comprises of animals, plants, and fungi. They are known to be much larger than prokaryotes which contain no nucleus because they are multinucleated organisms. Eukaryotes were said to have developed about 1.6 – 2.1 billion years ago. But that is only an approximation. We would not be here if eukaryotic microorganisms did not exist. These organisms tend to share a common origin and could be treated as a super kingdom, empire, or domain. Lastly, many unicellular organisms are also eukaryotes e.g protozoa. The types of eukaryotes are algae, yeast, fungi and protozoan.
Escherichia coli (E. coli) is a member of the Enterobacteriaceae family of organisms. It is a non-spore forming, facultative anaerobic, gram negative rod capable of growing on a variety of media and, similar to other members of the Enterobacteriaceae family, contains the enterobacterial common antigen. Most E. coli are part of the normal flora of the human gastrointestinal tract, however some strains are pathogenic and capable of causing clinical disease. Epidemiologic classification of E. coli is based on the expression of certain surface antigens. The three of greatest importance are the somatic O polysaccharide (part of the lipopolysaccharide or Gram negative endotoxin), the K antigens (part of the capsule), and the H antigens (flagellin proteins). The bacteria regulate the expression of these antigens through antigenic phase variation. This process allows the organism to selectively express or not express the antigens, which aids in protection from antibody-mediated cell death. Enterohemorrhagic E. coli (EHEC) are strains that produce exotoxins (particularly verotoxins) that result in hemorrhage of the intestinal mucosa. There are several serotypes of EHEC; the most clinically significant is O157:H7.
Lab Report 2: PGLO TRANSFORMATION EXPERIMENT Doris Daniels PURPOSE: The purpose is to transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar. Also, it is to observe if bacteria will grow in the presence of ampicillin and without ampicillin using two –DNA plates and two +DNA plates. INTRODUCTION: In this lab experiment, we will perform genetic transformation by putting some new DNA into E. coli cells.
Once the mixture had been completely dissolved, the solution was transferred to a separatory funnel. The solution was then extracted twice using 5.0 mL of 1 M
Saturated sodium chloride solution, also known as brine solution, is used to wash the distillate mixture. The distillate mixture is the phosphoric acid the co-distilled with the product. The brine solution also removes most of the water from the 4-methylcyclohexane layer. When six drops of 4-methylcyclohexene were added with two
Biology Lab Report Lab No. 18: Biochemical Genetics: Smooth Peas Wrinkled Peas Data Presentation: The diagram of cotyledon for smooth and wrinkled pea is attached to the next page. The table of starch presents is below: Type of Pea Starch Present? (Color change) Smooth