araC — gene that encodes the regulatory protein that binds to the pBAD promoter, only when arabinose binds to the AraC protein is the production of GFP switched on.
What role does the AraC protein play in the expression of GFP?
– So, in the presence of arabinose, araC protein promotes the binding of RNA to transcribe GFP.
What type of operon is AraC in the pGLO plasmid?
The pGlo plasmid has been modified to incorporate some features of the arabinose operon. Both the araPBAD promoter and the araC gene are present. The genes which code for araA, araB and araD have been replaced by the gene which codes for GFP.
What are the three important genes located on the pGLO plasmid?
This plasmid has been engineered to contain three core genes: the bla gene which encodes the enzyme β-lactamase, responsible for resistance toward the antibiotic ampicillin (AmpR), the green fluorescent protein (GFP) gene, originally derived from jellyfish (Aequorea victoria), which encodes the GFP, and, the arabinose …
What are the two genes in the pGLO plasmid and what is the function of each in the transformed E. coli?
coli is the pGLO plasmid, this plasmid contains the ampicillin resistance gene and the GFP gene. If the bacteria was able tosuccessfully be transformed with the plasmid, the bacteria will contain the two genes. The E. coli will be ampicillin resistant and will produce GFP.
What is the araC?
Abstract. The AraC protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric proteins. One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription.
CONTINUE READING BELOW
How is GFP expression induced or switched on during the transformation?
The gene for GFP can be switched on in transformed cells simply by adding the sugar arabinose to the cell’s nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates.
What is araC gene?
The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation).
How is pGLO plasmid made?
This recombinant plasmid, created by researchers at Bio-Rad, combines a gene for green fluorescent protein (GFP), cloned from a jellyfish, with control elements copied from a bacterial operon. The end result is a system that allows for bacterial expression of a eukaryotic gene.
Which pGLO gene triggers the GFP gene and fluorescence?
Arabinose activates araC and GFP genes which produce the fluorescence. [the arabinose promoter is activated (GFP gene) and then transcription is activated once the arc protein binds to the arabinose promoter.
What does the pGLO plasmid contain?
The pGLO plasmid contains an origin or replication, a selectable marker, and the gene for Green Fluorescent Protein (GFP). The plasmid also contains a gene for the arabinose C protein, which is a protein that regulates expression from the arabinose BAD promoter (PBAD).
What is the molecular mass of a pGLO plasmid?
Agarose vs.
In contrast the pGLO plasmid DNA has a molecular mass around 3300 kD (pGLO is 5371 base pairs long, and one b.p. of DNA has a molecular mass of 618 Daltons).
What are the extra traits or genes conferred to bacteria by the pGLO plasmid?
Meaning, the plasmid DNA, or pGLO, contains a gene for Green Fluorescent Protein (GFP) as well as the gene for resistance to the antibiotic ampicillin.
What does pGLO plasmid stand for?
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistance gene.
What organism is the sources of the pGLO gene in the transformation experiment?
The pGLO System
With the pGLO transformation kit, students use a simple procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria, and GFP causes the jellyfish to fluoresce and glow in the dark.
What does the GFP gene do?
Biologists use GFP as a marker protein. GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of the particular protein in an organic structure. Gfp refers to the gene that produces green fluorescent protein.
What does the AraC regulatory gene produce and where does it bind?
The regulatory gene, araC, is located upstream of the L-arabinose operon and encodes the arabinose-responsive regulatory protein AraC. Both araC and araBAD have a discrete promoter where RNA polymerase binds and initiates transcription.
How can the AraC protein function as both a transcription repressor and a transcription activator?
AraC Repressor and Activator
When AraC binds arabinose, the dimer changes configuration and binds to DNA at sites 1 and 2. Here, it acts as an activator, allowing the RNA polymerase to bind. C) When the repressor form of AraC binds DNA, it occupies sites 2 and 3, forming a loop in the DNA and causing gene inactivation.
How is the transcription of AraC genes turned off by the present of AraC?
The genes, araBAD and araC, are transcribed in opposite directions. … If arabinose is absent, the dimer AraC protein represses the structural gene by binding to araI1 and araO2 and the DNA forms a loop, which prevents RNA polymerase from binding to the promoter of the ara operon, thereby blocking transcription.
What is the purpose of the DNA transformation on the LB plate?
The purpose of this technique is to introduce a foreign plasmid into bacteria, the bacteria then amplifies the plasmid, making large quantities of it.
What is the purpose of CaCl2 in transformation experiment?
Calcium chloride heat-shock transformation is a powerful molecular biology technique used to introduce foreign DNA into a host cell. The concept of the technique is to render cells competent using CaCl2 to allow for introduction of plasmid.
How does arabinose interact with the araC protein help to turn on the RFP gene?
When arabinose is present, it binds to araC protein. RNA polymerase then binds to this complex &, can then transcribe the rfp gene.
What is the function of pBAD?
The pBAD expression system allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli.
How does the pBAD promoter work?
The PBAD promoter allows for tight regulation and control of a target gene in vivo. As explained above, PBAD is regulated by the addition and absence of arabinose. As tested, the promoter can be further repressed with reduced levels of cAMP through the addition of glucose.
Which of the following mechanism is operated by the AraC regulatory protein?
The AraC regulatory protein controls the transport and metabolism of the 5-carbon sugar arabinose. When arabinose binds to AraC, it converts it from a repressor to an activator.
How has pGLO plasmid been used for research?
In addition, pGLO incorporates a special gene regulation system that can be used to control expression of Green Fluorescent Protein (GFP) in transformed cells. … This glowing property is now used in research as a marker or tag to follow the movement of proteins within a cell in real time.
How was pGLO discovered?
In 1928, a England scientist called Frederick Griffith first discovered this technique, when he working with Streptococcus pneumoniae. Plasmid is a circular DNA molecule, which carrying one or more genes for antibiotic resistance proteins and GFP gene. … The procedure that use to transformed pGLO plasmid into the E.
Why does the pGLO plasmid glow on an LB amp ARA plate?
The pGLO plasmid has the bla gene which allows for ampicillin resistance. There are glowing colonies surrounded by satellite colonies, because ARA turns on the araC gene which turns on the expression of the GFP gene. When this is expressed in the presence of UV light, it fluoresces.
What is the purpose of the GFP gene in the pGLO plasmid quizlet?
pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin.
What is pGLO quizlet?
What is pGLO? a type of genetically engineered plasmid that encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin.
What are antibiotic resistance genes in the plasmid?
Plasmids are small DNA circles outside the bacterial chromosome. Several antibiotic resistance genes can be present on the same plasmid. In this example, they are called res A, res B and res C. Res A gives resistance to antibiotic A, res B to antibiotic B and so on.
How is genetic transformation related to gene expression?
Genetic transformation is a process that involves the introduction and expression of foreign genes in a host organism. This expression can result from the extrachromosomal, or episomal, presence of genes in nuclei that may persist if the introduced DNA has a mechanism for replication.
What is a plasmid select all that apply?
A plasmid is a piece of circular DNA present in bacterial cells that carries genes that confer new traits to a bacterial cell.
What is a DNA molecule used to carry a gene of interest from one organism to another?
recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.
How were restriction enzymes used in pGLO?
Restriction digests also form part of the foundation for molecular biology in general. Recombinant DNA molecules (such as the pGLO plasmid) are traditionally made by cutting various desired DNA fragments with restriction enzymes, then ligating (joining) the pieces together.
Do plasmids replicate?
The plasmid is a small DNA molecule within a chamber that is physically separated from chromosomal DNA and can replicate independently [6].
Which gene is responsible for making the bacteria resistant to ampicillin?
The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin.
What might explain the presence of nontransformed bacteria growing on the LB amp plate quizlet?
Explain your prediction. Bacteria which resemble the non-transformed will be found on the LB/(-) pGLO plate. These bacteria were removed from the starter plate, did not have any plasmid added to them, and were replated on an LB plate. Thus, they are virtually identical to the non-transformed starter.
How do the results of this experiment illustrate the relationship between DNA proteins and a trait?
How do the results of this experiment illustrate the relationship between DNA, proteins and a trait? DNA molecules code for proteins that help the cell function properly. The DNA is used to pass traits on to the offspring. … Very often an organism’s traits are influenced by a combination of its genes and its environment.