Introduction
Chitobiase, from Vibrio harveyi, is a membrane bound lipoprotein involved in the degradation of chitin. Chitobiase is similar to and may share a common ancestry to the a-chain of human b-hexos-aminidase. Chitobiase is encoded by chb.
In this experiment, a restriction map for restriction enzymes Eco R1, Pst1 and Hind III using Southern hybridization and restriction analysis of pRSG 192. pRSG 192 is a recombinant plasmid derived from the chb gene and pUC 19, a 2.7kb engineered plasmid which encodes for ampicillin resistance, a portion of the lac operon and a multiple cloning region . The chb gene exists as a 3.6 kb insert in the mutiple cloning region of pUC 19.
The major goals of Experiment One will be to isolate pRSG 192 from an overnight culture of E. coli, amplify a region of the chb gene using PCR, and to map restriction sites within the chb gene using restriction analysis and Southern hybridization.
Methods
Plasmid Isolation
Four microfuge tubes containing cell pellets representing 3.0ml of cells(2 x 1.5ml) from an overnight culture of E. coli were prepared. The supernatant fluid was discarded and each pellet was resuspended in 150ul of TE buffer(10mM Tris-HCl, pH 8.0; 0.1 EDTA). 300ul of SDS(1% SDS, 0.2 N NaOH) was added to each pellet. The tubes were placed on ice for five minutes, after which, 225ul of ice-cold 3M potassium acetate(pH 4.8) was added. The tubes were again placed on ice for five minutes and subsequently microfuged for five minutes.
The supernatants were recovered and transferred to new tubes. One volume of phenol/chloroform was added to each new tube. The tubes were shaken vigorously for two minutes and centrifuged for five minutes. The upper, aqueous phase was recovered and transferred to a new tube. One volume of chloroform was added to each tube. The tubes were vigorously mixed and microfuged for three minutes.
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
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
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
Firstly, an amount of 40.90 g of NaCl was weighed using electronic balance (Adventurer™, Ohaus) and later was placed in a 500 ml beaker. Then, 6.05 g of Tris base, followed by 10.00 g of CTAB and 3.70 g of EDTA were added into the beaker. After that, 400 ml of sterilized distilled water, sdH2O was poured into the beaker to dissolve the substances. Then, the solution was stirred using the magnetic stirrer until the solution become crystal clear for about 3 hours on a hotplate stirrer (Lab Tech® LMS-1003). After the solution become clear, it was cool down to room temperature. Later, the solution was poured into 500 ml sterilized bottle. The bottle then was fully wrapped with aluminium foil to avoid from light. Next, 1 mL of 2-mercaptoethanol-β-mercapto was added into fully covered bottle. Lastly, the volume of the solution in the bottle was added with sdH2O until it reaches 500 ml. The bottle was labelled accordingly and was stored on chemical working bench.
coli after addition of inducer IPTG at different times. We can see from the graph that it does not cut the X axis at time 0. This is due to the fact that the lac operon was not induced by IPTG due to lack of time. The graph cuts the X axis at roughly 1 and a half minutes after adding IPTG. This indicates the time where lac operon was first induced because there was beta-galactosidase produced. From here we can deduce that production of beta-galactosidase does not take place immediately after adding IPTG but rather takes a while for all the expression staged to be passed by lac operon in order to produce beta galactosidase. From the graph we can see that for the control no beta galactosidase was produced. This is because the control contains water and the repressor was allowed to bind to the operator, causing the transcription process to initiate due to RNA polymerase II not binding to the operator. There is a positive linear relationship between the time of induction with IPTG and the amount of beta-galactosidase production in the tubes. IPTG acts as an inducer, stopping the repressor protein to bind to the operator region by binding to the repressor protein, This causes the lac operon to be
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
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.
Next our group iced the tubes for ten minutes and labeled the plates with the materials that are inside of them, in this case +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB. Following the ten minutes on ice, the tubes were placed in water at 42 degrees Celsius for 50 seconds and then immediately moved back to the ice bath. After two more minutes on ice they were removed and, using the micropipetter with a new tip each time, 250 microliters of nutrient broth was added to each tube, they were then let to sit for ten minutes. When the ten minutes was up the tubes were gently flicked to agitate the solution and 100 microliters of the +pGLO tube was pipetted to each of the agar plates labeled
Purpose: The point of this lab was to determine whether or not DNA was actually extracted in the prior week’s experiment, in which E. Coli bacteria’s was lysed and through a series of chemical extractions it’s inner contents were harvested.
A recombinant plasmid are created by first using an enzyme that can identify and isolate specifically which gene that need to be cut. They are call restriction enzymes or restriction endonucleases, and more than 100 of these enzymes have been isolated. After the human gene (gene of interest) that codes for the desire trait is located on the chromosome restriction enzyme does it job, by cutting out the gene from the DNA. Now, the two ends of the human gene will be those that will link up with the open ends of the plasmid. An enzyme, DNA ligase, is used to couple each end of the gene to the open ends of the plasmid; this thus restores the circular DNA molecule with the human gene. Now the plasmid, with the human gene, is reinserted into the bacteria. They are then cultured and produced in large quantities of identical bacteria carrying the human gene. Now, these bacteria produce the human protein coded for by the spliced human gene. The protein is then isolated and purified and are ready to be injected into patients (crop, etc.) (Gish 1998).
Coli can in fact undergo mitosis on plates containing ampicillin, and show fluorescent qualities on a plate containing arabinose. These results logically follows from the fact that the plasmid inserted possesses qualities that allow for ampicillin to be broken down, and therefore not harm the E. Coli, meaning growth on plates containing ampicillin is proof of genetic transformation. Similarly, transformation of the E. Coli is also evident on the plate containing arabinose, as there was not only growth, but clear fluorescence under the blacklight, as the plasmid also codes for that expression. It is clear that those results are a result of the plasmid, as the plates treated with the +pGLO solution can be compared to those with the -pGLO solution, in which there was no growth on any plate except for the LB broth plate. Growth on the LB plate indicates that the E. Coli is healthy, and capable of mitosis in certain conditions, but lack of growth on the other plates points to it still being wild type. Therefore, it is clear that the +pGLO E. Coli have adopted new genes that allow for new functions that wild type E. Coli are incapable of, in addition to showing that the genes were transcripted and translated in S phase of mitosis, as daughter cells possess similar qualities, as shown by their ability to subsequently grow and divide. This In further examining the plates treated with the +pGLO solution,
In this experiment the bacteria E. Coli will be genetically transformed into a competent by going through a process called Heat shock. Heat shock is when you take a bacterial cell and have sudden increase in temperature which increases the permeability of the plasma membrane this causes the cell to take up the DNA from the surrounding medium. (Lab Manual) There are several other methods of genetic transformation but in this lab those will not matter. In this specific experiment pGLO will be the medium around the E. Coli. Genetic transformation is the active up take of foreign DNA in a bacterial cell. (PubMed) For genetic transformation to occur we have to have a medium that contains a different DNA than the thing we are trying to genetically transform. The medium in this experiment will be the plasmid pGLO. This pGLO plasmid is a vector, which transfers a gene from one organism to another. (Lab Manual) The plasmid contains the GFP (green fluorescent protein) gene which makes the bacteria glow in the presence of a sugar called arabinose. The pGLO also contains a gene for resistance to the antibiotic ampicillin. Ampicillin will be used to see if the bacteria lived because the ampicillin is an
...Brighter appearance to coloured textiles thanks to a new cellulase from an extremophilic bacterium. Journal of Biotechnology 66, 231–233.
In this method, living spores which are resistant to whichever sterilizing agent is being tested are prepared in either a self contained system, such as dry sp...