How was GFP cloned?
The GFP was cloned into pProEx HTb by digesting the pAVA319 with Nco I and Xba I. The resultant Pro319 was transformed into JM109.
What is the first cloning vector?
plasmid PBR322
What vectors are commonly used for cloning?
The most commonly used cloning vectors are E. coli plasmids, small circular DNA molecules that include three functional regions: (1) an origin of replication, (2) a drug-resistance gene, and (3) a region where DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene.
What vectors are used for EGFP?
Vectors. pEGFP-N1 (Clontech Laboratories, Inc. GenBank Accession #U55762) vector containing GFP gene, and pCDNA3. 1/ His/ LacZ vector (Invitrogen life technologies) which includes neomycin resistant gene, were used in these experiments.
How many base pairs is GFP?
Most commercially available genes for GFP and similar fluorescent proteins are around 730 base-pairs long. The natural protein has 238 amino acids. Its molecular mass is 27 kD.
Is GFP a vector?
Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it emits green fluorescence after blue-light excitation. Inclusion of this gene in a vector can allow rapid selection of successfully transduced cells.
Can GFP enter cells?
Because of its small size, GFP can enter nuclear pores, so that GFP not fused to other polypeptides is found in both cytoplasm and nucleus in plant, animal and yeast cells (Grebenok et al., 1997a, b; Köhler et al., 1997b; Haseloff and Siemering, 1998; Fig. 1A).
How is GFP produced?
Gfp refers to the gene that produces green fluorescent protein. Using DNA recombinant technology, scientists combine the Gfp gene to a another gene that produces a protein that they want to study, and then they insert the complex into a cell.
What is wild type GFP?
Wild type GFP from jellyfish displays an absorption spectrum with two peaks, a major one at 395 and a minor one at 475 nm, with extinction coefficient of 30000 and 7000 M−1 cm−1, respectively. GFP fluorescence originates from a Ser-Tyr-Gly-sequence that forms a p-hydroxybenzylidene-imidazolidone.
Is GFP toxic to cells?
In addition to initiating the apoptosis cascade, reactive oxygen production induced by GFP has been linked to cellular toxicity and eventual death in GFP expressing cells.
Why is EGFP better than GFP?
EGFP is brighter and matures rapidly at 37°C than wild-type GFP [1, 9]. Protein engineering of EGFP has yielded several green variants with improved characteristics such as Emerald. This Emerald FP has improved photostability and brightness than EGFP [11].
Is GFP a reporter gene?
Since the cloning and enhancement of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria (4, 7, 9, 27–29, 41, 46), GFP has been widely used as a reporter gene.
How does GFP work as a reporter gene?
Green fluorescent protein (GFP) causes cells that express it to glow green under UV light. A specialized microscope is required to see individual cells. Yellow and red versions are also available, allowing the investigation of multiple genes at once. It is commonly used to measure gene expression.
How is a GFP reporter gene useful?
Reporter genes were described in the previous chapter as useful markers to localize protein expression. Fusing a fluorescent reporter gene, such as GFP or its variants, to a protein of interest allows a scientist to observe the location and trafficking of the protein in live cells and tissues (Figure 7.7).
Why is Luciferase the best reporter?
Luciferase-based reporter gene assays are widely used because of their ultrasensitive detection capacity and wide dynamic range.
What is the advantage of using luciferase as a reporter instead of GFP?
As a result of the different mechanisms to produce photons, chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage of lower background levels and improved signal sensitivity since photons are simply measured – they are not required to initiate the reaction.
What does luciferase bind to?
In this organism, the luciferase (Renilla-luciferin 2-monooxygenase) is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein.