Lab Partners : Sage Gibson And Vanessa Song

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

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

Next, the transformation efficiency was calculated by dividing the total amount of DNA on the agar plate in μl by the number of cells growing on the +pGLO LB/amp/ara plate.

One of the most imperative functions in maintaining the development of evolution is the frequency of genetic transformation: the injection of foreign DNA into another organism’s DNA. This term is defined by the actions of a vector, but more specifically by the actions of plasmids and phages. However, in this experiment we are primarily focused on the effect of the pGLO plasmid transformation of GFP on the E. coli bacteria by introducing a second chromosome or a plethora of cloned plasmids. (Bassiri 2011)

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).

The reason why it did not inhabit any change after the experiment was conducted is because it had no plasmids added to support the growth and the ampicillin actually killed the bacteria. The plasmid would have served as the vector to insert the gene into the E. coli. The -pGLO LB plate thrived because there was no ampicillin present. While the +pGLO LB/amp/ara plate ended up glowing under the UV light because the GFP was only able to show with the presence of the arabinose, because it is what activates the operon. The +pglo LB/amp plate contained a few colonies but it did not glow under the UV light due to the absence of the

After that, a new sterile loop was used to immerse the pGLO plasmid DNA stock tube into the +pGLO tube but no into the – pGLO tube. Both tubes were well closed and put back on the ice for 10 minutes. While both tubes were sitting on ice, the four plates were labeled. LB/amp plate was labeled +pGLO, LB/amp/ara plates were labeled +pGLO, LB/amp plate was labeled – pGLO, and LB plate was labeled – pGLO. After that, the tubes were transferred into a water bath set at 42° C for exactly 50 seconds, then placed rapidly back on the ice for another two

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The goal of this experiment was to create a “super e. coli,” as mentioned in the introduction, by co-transforming the pKan and pGlo plasmids. Since the e. coli were not able to absorb both plasmids at the same time, it can be noted that the transformation did not work. The plates containing the pKan and pGlo plasmids separately demonstrated a successful transformation, indicating the bacteria’s inability to take in two plasmids at the same time. Both of these plates acted as our controls, so in the end we could

The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The plasmid that was inserted into the E. coli culture had antibiotic-reistance gene and a reporter gene. A reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the DNA to

In this lab the genetic process known as transformation will be used to make E. coli bacteria cells glow in the dark by adding GFP. Transformation is when a cell’s genotype is changed by adding exogenous DNA. This process results in the cell accepting the added genes and incorporating them into the reproduction process, creating more cells with the new gene. Genetic transformation naturally occurs in some species of bacteria, however in this lab it will be generated artificially (Bacterial Transformation). In order for transformation to take place the bacteria must be competent. This means that the bacterium needs to be ready to take in the added genetic information from its environment. These genes are absorbed through the cell membrane (Genetic Competence in Bacillus Subtilis). However, for transformation to occur, a restriction enzyme is required.

The plasmid used is called the pGLO plasmid, which has been genetically engineered. The pGLO plasmid carries the GFP gene. When GFP is produced, the transformed bacterial colonies will glow bright green under UV light. Green fluorescent protein (GFP) is the trait we are primarily looking for in this experiment. For growth, the positive control was the -pGLO LB dish because it had nothing to inhibit its growth, and the negative control was the -pGLO LB/ampicillin dish, because it did not have the genes for Beta-Lactamase to protect it from ampicillin, so it could not grow.

The objective of this experiment was to transform E.coli into having genes resistant for ampicillin and kanamycin by using recombinant plasmids. The three steps of the experiment include ligation, transformation, and growth on media. Restriction enzymes BamHI and HindIII splice the DNA. The recombinant plasmid is formed and combined with E.coli. Four experimental plates and four control plates were incubated at 37 degrees Celsius for 24 hours, then examined for growth. Lawns of E.coli grew on the LB plates. The

The LB/amp- plasmid petri dish had no growth because ampicillin was incorporated. Ampicillin is an antibiotic that kills potential growth for a bacteria. The LB-Plasmid petri dish eventually grew a lawn of bacteria, this dish only had Laurel broth which is food for bacteria, explaining the substantial growth. We observed circular colonies that only grew on a little corner of the dish. This awkward growth is probably due to the inaccurate spreading of the cells.The LB/amp+plasmid petri dish had growth as well because the ampicillin resistance gene was able to grow since the plasmid contained a gene allowing for antibiotic resistance. The LB+plasmid petri dish had growth because there was no antibiotic to inhibit

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