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Recombinant dna technology campbell
Recombinant dna technology and biotechnology
16.4 Use of recombinant DNA technology
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Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping. The aim of this experiment was to isolate cDNA molecule CIH-1 (Colletotrichum lindemuthianum CIH1 gene) that is contained in vector pBK-CMV and transfer it into cloning vector pUC19. This was attempted by conducting a restriction digest of vectors pUC19 and pBk-CMV containing CIH-1, using restriction endonucleases Xba1 and EcoR1 and the characterization of recombinant plasmids. 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). The restriction digest was performed using restricti... ... middle of paper ... ...but that it is too small to be seen. The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants. Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
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 two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
In order to figure out the genes responsible, there are several other factors that must be determined. These factors include the number of genes involved, if each gene is x-linked or autosomal, if the mutant or wild-type allele for each is dominant, and if genes are linked or on different chromosomes. Proposed crosses include reciprocal crosses between the pure-breeding mutants of strains A and B with the wild-type will help determine if the genes or sex-linked or autosomal, in addition to which alleles are dominant (8). Another proposed cross includes complementation crosses between pure-breading mutants from strains A and B to determine if one or two genes are involved (8). Furthermore, testcrosses between F1 progeny and pure-breeding recessive mutants from strains A and B, which will help determine if genes are linked on the chromosome or if they assort independently (8). These proposed crosses are shown in the attached
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).
New research techniques have made it possible to engineer and explore differences in the sets of chromosomes in organisms. This has been a technological revolution during the last decade. Allowing scientists to be able to explore DNA to a new extent. During the process of this research it has come apparent that foreign DNA inserted into self-replicating genetic elements such as bacteria plasmids can replicate. This breakthrough has also shown that the plasmids that have been used can also be used to change the genetic constitution of other organisms (1).
Holton, T. A., and M. W. Graham. "A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors." Nucleic acids research 19.5 (1991): 1156.
...thin pTrc99A/topAcysB plasmid, then the primers would not adhere to the exclusive sites found within topAcysB gene and the ligation of this product into M13mpI9 would not happen. To verify whether topAcysB was cloned, the multiple cloning site embedded in the lacZ ' gene of M13mp19 prevents alpha-complementation when DNA is cloned into one of the restriction sites.(1) Therefore, a blue plaque on media forms when M13mpI9/topAcysB is not ligated successfully, and white plaques will appear if M13mpI9/topAcysB was ligated successfully. The plates retrieved from this procedure exhibited white plaques, data not shown in the results, but having white plaques present, illustrates that topA gene had been cloned through PCR. The pTrc99A/topAcysB plasmid has been proven to be a useful expression vector, and has multiple applications in the process of forming topoisomerase I.
In this experiment, the bacteria, E Coli, was transformed with the Green Fluorescent Protein (GFP). To start, the bacteria was grown, harvested, and added to a tube with -DNA. Calcium chloride was also added to the tube to allow the cells to take up the DNA and become transformed. Half of this mixture was then placed into a tube with +DNA, which contains the Green Fluorescent Protein, and both were placed in an ice bath for 10 minutes. When time was up, the tubes were placed in 42℃ water for 90 seconds. Next, the tubes were put back in ice water for 2 minutes. Then recovery broth was added to give the bacteria more fluid to grow in and provide nutrients, and the tubes were placed into 37℃ water for 15 minutes. The purpose of placing the tubes in warm and cold water was to heat shock the mixture so the cell membranes would become permeable and the DNA could enter the cells. Once the heat shocking was complete, the -DNA and +DNA were placed on 4 petri dishes with agar. The -DNA was placed on one dish with only agar, and another dish with
Once plasmids are digested and confirmed the next phase of making recombinant DNA is to successfully ligate the fragments. Ligation is the process of sealing sticky ends of plasmids fragments that contain the ampicillin resistant gene or the kanamycin resistant gene. Refer back to Figure 3 for a visual representation of ligation in action. Once ligase is added to the sample, a confirmation test must be done in order to prove ligation successfully occurred. One must remember that ligated plasmids will be enormous. The reasoning being is that, the fragments that were cut by the restriction enzyme where big. Therefore, these
Discoveries in DNA, cell biology, evolution, and biotechnology have been among the major achievements in biology over the past 200 years with accelerated discoveries and insight’s over the last 50 years. Consider the progress we have made in these areas of human knowledge. Present at least three of the discoveries you find to be the most important and describe their significance to society, heath, and the culture of modern life.
There are two type of transduction which is generalized and specialized transduction. Generalized transduction is where phages can carry any host gene while specialized transduction only specific host gene can be transferred by transducing phage (3,4). This process was discovered by Lederberg and Zinder in 1952 while they were studying the process of conjugation in Salmonella typhimurium using same method that was used in determination of E coli conjugation method. Two different strains were used which phe− trp− tyr− in one strain and the other was met− his− (3,4). There are no wild-type cell observed after the strains was plated individually, however when the two strain were combined wild-type strain become visible at about 1 in 105 frequency (3). Thus they conclude that the situation is similar to the recombination of E. coli and therefore E.coli also used transduction as their mating process. Interestingly, in 1970, Morton and Akiko Higa confirmed that transformation can also occur in E.coli using artificial method of
It stands to hypothetical reasoning, that If bacteria with +pGLO plasmids that are resistant to the antibiotic ampicillin and have the gene for GFP, colonies will survive and grow on the transformation plates that have LB/amp. In addition, +pGLO bacteria on a plate with LB/amp/ara will grow and glow green under ultraviolet light because of the inclusion of arabinose which switches on GFP. In the control plates, -pGLO
The discovery and characterization of restriction enzymes first took place in the late 1960’s and early 1970’s. The scientists responsible for the discovery were molecular biologists Werner Arber, Hamilton Smith and Daniel Nathans. In the late 60’s Arber observed a sharp change in the bacteriophage DNA he had been working with after it invaded resistant strains of bacteria. It had been cut into pieces and degraded. He hypothesized that bacteria could express two different enzymes: one that recognizes and destroys foreign DNA, the restriction enzyme, and one that modifies bacterial DNA to protect it from the former, a modification enzyme. A short time later he, along with Stuart Linn, confirmed his second hypothesis that both enzymes act on the same specific sequence of DNA, the recognition sequence. In 1970 Hamilton Smith both verified and elaborated on Arber and Linn’s hypothesis and initial discovery using a
Recombinant DNA technology has opened the door for humans to isolate and purify virtually any known genomic sequence. The human genome is known to contain approximately 6x109 base pairs over a span of 23 pairs of chromosomes. Getting pure DNA samples from large genomes like ours is now made far easier thanks to recombinant DNA technology. In addition, functional regions can be investigated and studied in predetermined manners, giving us vital insight to the biochemical, molecular, and genetic properties of our DNA (Lodish et al., 2000). Recombinant DNA itself is any DNA molecule formed by joining DNA fragments from different sources. The most frequent manner that recombinant DNA is produced is by restriction digestion, followed by ligation of the complementary sticky ends via DNA ligase. Each step in the creation of the construct plays a vital role, and should not go unrecognized.