The green fluorescent protein (GFP) gene is a naturally occurring gene from a bioluminescent jellyfish. The gene allows for objects and animals to glow in the dark when activated by the presence of the sugar arabinose in the pGLO plasmid. The GFP gene is often used as a marker for gene expression and genetic transformation. The pGLO plasmid is a genetically engineered plasmid used as a vector in biotechnology to generate genetically modified organisms(GMO). M. Chalfie et. al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. 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 Another weakness being not effectively utilizing the heat shock method of genetic transformation, the transformation solution of calcium chloride could not have gotten cold enough when sitting on the ice bath for certain periods of time. This lowers the effectiveness of heat shock genetic transformation as the plasma membrane of the cells do not become permeable enough to be able to take up the foreign DNA that it is being exposed
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.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
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.
...lasmid have the capability to survive, and multiple in number as they expand and reproduce. In addition, restriction enzymes have led to gateway discoveries in the topic of cloning. Essentially, because these restriction enzymes have allowed for the removal of a fragment of DNA and for it to be placed in another location, this idea has led to scientists being able to integrate exogenous DNA into natural plasmids that may ultimately lead to cloning plasmid vectors. These plasmids then have the ability to self-replicate (neb.com). The discoveries made surrounding these restriction enzymes have paved the way for the cloning of DNA. Furthermore, DNA mapping is a practical application stemming from restriction analysis that now allows for scientists to be able to detect insertions and deletions, single nucleotide polymorphisms, and identifying genetic disorders (neb.com)
First a control was established for E. coli in a 1.0x nutrient broth. This was
(2005). Anti-diarrheal effect of Galla Chinensis on the Escherichia coli heat-labile enterotoxin and ganglioside interaction. Journal of Ethnopharmacology, 385-391.
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
This biotechnology lab analyzes the effect of transferring genetic information through the alternation of bacterial gene in E. coli (Spilios, 2014). This alteration occurs through plasmid DNA transcribing the new genetic components into RNA, which will translate into an amino acid (Sadava et al., 2014). This newly transcribed amino acid is an enzyme that will give the transformed E. coli cells an antibiotic resistance, Beta-lactamase (Greenfield et al., 2009). The plasmid DNA of interest will be altered to become more resilient to the antibiotic ampicillin, since beta-lactamase could decompose the ampicillin. In addition to plasmid DNA, the bacteria contain other important features such as reporter gene. This reporter gene will act as an aid when observing the effect of the alteration, since this particular gene can be distinguished when a plasmid with foreign DNA is transferred from one to another (Spilios, 2014). Moreover, the reporter gene being used in this lab, Green Fluorescent Protein, is to determine gene resistance to ampicillin. GFP would be useful in this experiment, since it would glow when arabinose operon is present. Ampicillin is a derivative of penicillin that inhibits bacterial growth by interfering with the synthesis of bacterial cell walls. Since E. coli is gram negative, and ampicillin kills the gram-negative bacteria by synthesizing with the cell wall, E. coli should perish under no transformation. However, the ampicillin resistance gene is the enzyme Beta-lactamase, which is secreted by transformed cells into the surrounding medium where it destroys ampicillin (Dörr, 2010). In order to resist ampicillins, E.coli utilizes pGLO plasmid to protect the cell from ampicillin’s invasion. There are four components to...
Bacterial growth may be controlled by many methods; the techniques relevant to this experiment include heat, ultraviolet (UV) light, and antimicrobial control. Using heat as a means of controlling bacterial growth is favorable because it is quick, safe, and cost-effective (Nester, 2007). There are two kinds of heat: moist heat, which destroys the proteins of microorganisms by boiling or steaming, and dry heat, which requires high temperatures to oxidize cell components and damage proteins by incineration or dry heat ovens (Nester, 2007). Cellular proteins are essential in carrying out important biological activities, so without them, the bacteria will not be able to survive (Nester, 2007). Moist heat is widely used to treat drinking water,
A lysozyme was used to break down walls of the bacteria to allow the RFP to be released. It was hypothesized that after the solution was put through the HIC column, the RFP would be isolated from the bacteria. The HIC matrix binds to the hydrophobic molecules of the protein in high salt conditions. Red fluorescent protein is hydrophobic while bacterial proteins are not. The bacterial proteins will pass through the column while RFP will stay in the column.
“Andi” is a backward acronym for “inserted DNA” that describes the method used by scientists at the Oregon Regional Primate Research Center (ORPRC) in Beaverton. The lead scientists Gerald P. Schatten and Anthony W.S. Chan, along with their team, placed copies of the green fluorescent protein (GFP), found in jellyfish, in specialized viri: retroviri. Their main goal was to create a monkey with a new gene introduced in a laboratory, thus a transgenic monkey. The significance of the GFP gene was to provide quick, detectable, and vivid evidence of whether the experiment was successful. These “replication-defe...
All the bacteria on the petri dish took in the plasmid and are therefore alive. The gene did not glow because Arabinose was not present and they could not turn on the operon. LB/ Amp /Ara with pglo glowed because the arabinose turned on the operon that controlled glowing. The bacteria is now making arabinose, causing them to glow. Also, all the bacteria on the petri dish were resistant to Ampicillin. Some of the bacteria colonies that did not glow had metabolized, or used up, all the arabinose. Bacterial transformation has shown great promise and progress in medicine and agriculture. It helps with research of using Insulin to treat diabetics, creating, and inserting cell hosts. Botanists use bacterial transformation to experiment with plants to resist colder temperatures, playing with the ripening process, and their ability to form a resistance to pests. However, these are always contradicted with statements challenging the ethical and moral issues that have to be unravelled with much thought before the further use of bacterial transformation. This process has opened doors for a healthier future and new, easier ways to create life saving
A GMO is a plant or animal that has been genetically engineered with DNA from bacteria, viruses, or other plants and animals. Most of the combinations which are used could not possibly occur in nature on its own. The intention of the process is to create a new beneficial trait such as creating its own pesticide or make it immune to herbicides. This would allow the crop such as Bt co...
In this day and age, Genetically Modified Organisms (GMOs) have become a topic of large interest in the media. GMOs are defined as an organism whose genetic structure has been altered by incorporating a gene that will express a desirable trait (Dresbach et al. al. 2013). Often times, these traits that are selected are either beneficial to the consumer or producer. Currently, GMOs are being created at a higher rate than ever before and are being used in the foods that we eat.
LAB REPORT 1st Experiment done in class Introduction: Agarose gel electrophoresis separates molecules by their size, shape, and charge. Biomolecules such as DNA, RNA and proteins, are some examples. Buffered samples such as glycerol and glucose are loaded into a gel. An electrical current is placed across the gel.