BME-603 Exam 2 - Tao Xu (Nov-9-2016)
Origin of replication is the starting point of DNA replication. It cannot replicate in the host cells without this site.
Antibiotic resistance gene can be served as a select agent to find the right clone and then improve the plasmid transformation efficiency. Antibiotic resistance gene can also give the bacteria a pressure to have the plasmid, because the bacteria would use more energy to replicate both the plasmid and its own DNA.
(1) The protein needs complex eukaryotic post-translational modifications for structure.
(2) The eukaryotic proteins are not well folded in E. coli and form insoluble aggregates.
(3) Proteins are secreted, membrane or intracellular.
cDNA library is usually used to expressing eukaryotic gene in prokaryotes. There are no introns in E. coli DNA, so it does not have enzymes which could cut it out in the transcription procedure. In cDNA library, there are no introns, so it could be expressed in E. coli cells.
Genomic DNA library have the non-coding and regulatory sequence while cDNA library doesn’t, but genomic DNA library is hard to make and maintain.
(1) 2-10 kb fragments: Plasmid
Reason: Plasmid is autonomous and self-replicating circular extra-chromosomal DNA molecule. The plasmid-based vectors is usual used to clone small DNA inserts up to 15 kb.
(2) 15-20 kb fragments: Bacteriophage lambda
Reason: Bacteriophage λ is an E. coli virus. Insertion vector is usual used to clone inserts from 5–11 kb, while
3) As a ribosome moves along the mRNA, the genetic message is translated into a protein with a specific amino acid sequence.
In order to find transformation efficiency, the total number of colonies on the plate is divided by the total amount (µg) of DNA spread on the plate. All the factors that must be taken into account while finding the transformation efficiency include; the total amount (µg) of plasmid DNA used, the total volume (µl) of cell suspension prepared, the fraction of DNA spread on the plate and finally the total amount (µg) of DNA present on the plate. In the LB/AMPC plate the transformation efficiency was 8.2 x 103 colonies per µg of plasmid DNA while the LB/AMPlux plate the transformation efficiency was 1.07 x 104 colonies per µg of plasmid DNA. The transformation efficiency for the LBC and LBlux plates were not taken into account, even though they too transformed because they were not in a restrictive environment where they needed to express their ampicillin resistance genes in order to
134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).
First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.
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).
With two T7 promoters, the PCR product will be transcribed by the T7 polymerase. Therefore, a double stranded RNA (dsRNA) product is produced from the plasmid, once IPTG is added to activate the lac operator of T7 polymerase.
For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).
Certain E. Coli strains are also known to show resistance to bacteria killing antibiotics. This resistance is due to the plasmids, or small round DNA molecules, in the bacteria that carry the resistant genes. R Plasmids (resistance plasmids) are widely studied and bestow resistance to factors that inhibit growth of the organism. R plasmids code for proteins that can
Genetic engineering is used in health treatments, agricultural applications, and environmental solutions. Genetic transformations incorporate foreign genetic material into the DNA of a different organism via a vector, which carries the genetic material. Plasmid DNA is small, round, and autonomous, due to its origin of replication. In biotechnology, plasmids carry beneficial genes, such as antibiotic resistance, and also a reporter protein, in this case, Green Fluorescent Protein
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].
DNA replication is described as semi-conservative. It is semi-conservative because the replication of one helix results in two daughter helices each of which contains one of the original parental helical strands. Furthermore, it is semi-conservative because the two new daughter DNA molecules are “half old” and “half new”; this means that half the original DNA molecule is saved, or conserved in the daughter DNA molecules.
These new formations are held together by hydrogen bonds. The third level is the tertiary structure. The tertiary structure of a protein is a contorted secondary structure being twisted and folded all out of shape to form a 3-d complex. The type of bonding that holds these formations together are weak interactions such as hydrophilic, hydrophobic, ionic, and hydrogen bonds. These bonds are individually weak, but collectively strong. The forth level, which completes a protein, is quaternary structure, which occurs when two or more tertiary structures are joined together by polypeptide bonds.
Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial
The process of DNA replication plays a crucial role in providing genetic continuity from one generation to the next. Knowledge of the structure of DNA began with the discovery of nucleic acids in 1869. In 1952, an accurate model of the DNA molecule was presented, thanks to the work of Rosalind Franklin, James Watson, and Francis Crick. To reproduce, a cell must copy and transmit its genetic information (DNA) to all of its progeny. To do so, DNA replicates following the process of semi-conservative replication. Two strands of DNA are obtained from one, having produced two daughter molecules that are identical to one another and to the parent molecule. This essay reviews the three stages