Subcloning Of Fungal CDNA From PBK Lab

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

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).

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).

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

The bacteria has ampicillin resistance gene and can either have no RNAi insert (control plate) or PCR product that is a portion of target gene (unc-22).

The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

For this experiment, we used two restriction enzymes called Ava II and Pvu II. Four tubes were needed for the experiment as well. Each microtubule was labeled accordingly, A, B, and C. Using Micropipettes, we added 2 microliters of pUC19 DNA into each tube. To make sure the DNA was on the bottom of the tube, we tapped each tube on the lab bench. Each tube had its own specific amount of different solutions added on, however the final volume for each tube was 30 microliters. Tube A had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Ava II enzyme. Tube B had, 24 microliters of DI water, 3 microliters of the buffer, and 1 microliter of the Pvu II enzyme. Tube C had, 23 microliters of DI Water, 3 microliters of the buffer, 1 microliter of Ava II, and 1 microliter of Pvu II. The last tube C had, 25 microliters of DI water, 3 microliter of the buffer, and no enzymes. Each solution was added according to how it is written here. After we finished adding the solutions to each tube, we tapped the tubes on the lab bench to make sure the

This experiment was performed to assess the efficacy of genetic transformations on bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and placing them on various media to observe gene expression via growth and color under UV light. The transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of arabinose, the transformed E. coli glowed green. These results support the previous scientific understanding of bacterial competency, vectors, and gene expression and support gene transformations as an effective method to transfer the desirable DNA of one organism into another organism’s DNA. These results can be applied to real world issues such as medical treatments, food production, and environmental conservation.

The transposon in this experiment is contains kanR in between the inverted repeats on either end, which will be transposed from the plasmid pVJT128 to the chromosome of the recipient bacteria.

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].

In this laboratory experiment, we was introduce to an introduction to streaking and spreading of bacteria in agar plates such that single cells can be isolated from one another, each cell can reproduces to form a visible colony composed of genetically identical clones. Streaking and spreading bacteria is to obtain individual colonies is usually the first step in genetic manipulation of microorganisms.

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

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double