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
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
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
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...
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)
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...
First a control was established for E. coli in a 1.0x nutrient broth. This was
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,
(2005). Anti-diarrheal effect of Galla Chinensis on the Escherichia coli heat-labile enterotoxin and ganglioside interaction. Journal of Ethnopharmacology, 385-391.
“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...
The purpose of the lab was to transform E.coli using the plasmid pRFP to promote the expression of antibiotic resistance as well as expression of the red fluorescent protein (RFP). The hypothesis was that if the transformation was successful, then the bacteria would express RFP because the arabinose would activate the plasmid’s red fluorescent protein, and show growth because pRFP allows E.coli to grow even in the presence of an antibiotic. The plasmid was combined with a sample of E.coli through the process of transformation. Following transformation, the protein was isolated using hydrophobic interaction chromatography (HIC) and the size was determined using SDS-PAGE. The results showed that the E.coli transformed by the pRFP would thrive and express the RFP in the proper environment.
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
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.
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.