pET 3A Vector:
- Double-stranded DNA separation or denaturation.
- Primer annealing to template DNA.
- Primer extension.
PCR Extender enzyme:
- Phytochrome-like proteins found in non-photosynthetic eubacteria, which functions as a light-regulated histidine kinase, which helps protect the bacterium from visible light.
Source: Davis, S. J., A. V. Vener, et al. (1999). "Bacteriophytochromes: phytochrome-like photoreceptors from non-photosynthetic eubacteria." Science 286(5449): 2517-20.
- open-chain tetrapyrroles appearing as intermediates in the heme degradation pathway, play important roles in biological complexes. They serve as chromophores in light harvesting systems in cyanobacteria and are chromophores in light-sensing photoreceptors of the phytochrome type.
Source: Acc Chem. Res. 2010 Apr 20; 43(4):485-95.The role of the chromophore in the biological photoreceptor phytochrome: an approach using chemically synthesized tetrapyrroles.Bongards C, Gärtner W.
- An enzyme which catalyses the oxidative degradation of heme to yield equimolar amounts of biliverdin (BV), iron, and Carbon monoxide.
In the well-studied eukaryotic Heme oxygenases (HOs), NADPH and NADPH-Cytochrome P450 reductase serve as the electron donor. Bacterial HOs have only been recently discovered. For example, in C.diphtheriae the HO reaction is utilised to release iron from heme under pathogenic (free-iron limiting) conditions. In fact, the breakdown of heme to mine iron is thought to be the major function of HOs from pathogenic organisms, thus allowing them to overcome the low concentration of free-iron necessary for successful colonisation. Furthermore, bacterial HOs are also involved in the first step of phycobilin biosynthesis. These linear tetrapyrrole molecules are precursors of the chromophores for the cyanobacterial light-harvesting phycobiliproteins and the photoreceptor phytochrome. Phytochromes are traditionally known as biliprotein photoreceptors in plants but have recently also been discovered in bacteria. Unlike plant and cyanobacterial phytochromes, which carry a phytochromobilin or phycocyanobilin chromophore, bacteriophytochrome (BphPs) from non-photosynthetic prokaryotes have been shown to utilise a BV chromophore.
Source: Rosalina Wegele, Ronja Tasler,Yahong Zeng, Mario Rivea and Nicole Frankenburg-Dinkel (2004) October 29, “The Heme Oxygenase(s) Phytochrome System of Pseudomonas areuginosa” The journal of Biological Chemistry, 279, 45791-45802.
- A vector is a plasmid that allows cloning of target DNAs at sites where they will be minimally transcribed by E.Coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase in E.Coli cells. pET vectors stand for: plasmid for Expression by T7 RNA polymerase. Numbered suffixes denote different upstream or downstream configurations, such as different restriction sites. Vectors containing the T7 promoter directly ahead of a unique cloning site are referred to as transcription vectors, and have a number suffix.
Transcription vectors can also be used for translation if the target DNA carries its own translation signals. Vectors that carry both the T7 promoter and translation start signals ahead of the cloning site are referred to as translation vectors, and have an additional letter suffix. The letter suffix indicates which triplet of the Bam H1 cloning site (GGATCC) is in the correct reading frame for translation, ‘a’ for GGA, ‘b’ for GAT, and ‘C’ for ATC.
Source: Rosenberg, A. H., B. N. Lade, et al. (1987). "Vectors for selective expression of cloned DNAs by T7 RNA polymerase." Gene 56(1): 125-35.
- a detailed description of the order of the chemical building blocks, or bases, in a given stretch of DNA.
- This is an umbrella term traditionally used by scientist to describe different processes for duplicating biological material. There are different types of cloning which can be categorised under the following three cloning technologies: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.
The terms “recombinant DNA technology,” “DNA cloning,” “molecular cloning,” and “gene cloning” all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell.
Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal.
Therapeutic cloning, also called “embryo cloning,” is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. These stem cells can be used to generate virtually any type of specialised cell in the human body. Stem cells are extracted from the egg after it has divided for five days. The extraction process destroys the embryo, which raises a variety of ethical questions.
- Transformation (Bacterial transformation) is the process by which bacterial cells take up naked DNA molecules. Bacteria, which are able to uptake DNA, are called “competent” and are made so by treatment with calcium chloride in the early log phase of growth. The bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions.
As the chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cell to swell and is necessary for the uptake of DNA. The exact mechanism of this uptake is unknown. It is known, however, that the calcium chloride treatment be followed by heat. When E.coli are subjected to extreme heat, a set of genes are expressed which aid the bacteria in surviving at such temperatures. These set of genes are called heat shock genes. The heat shock step is necessary for the uptake of DNA.
- PCR stands for Polymerase Chain Reaction, which is a technique for copying a piece of DNA a billion fold. The process creates a chain of many pieces, in this case, the pieces are nucleotides, and the chain is a strand of DNA.
PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme’s ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) derived from thermophilic bacteria.
Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or “primers” to get the DNA polymerase started. Broadly speaking, there are three steps identified by incubating at different temperatures, the three steps make up a PCR “cycle”.
- A specialised polymerase enzyme, which can synthesize a complementary strand to a given DNA strand in a mixture containing the 4 DNA bases and 2 DNA fragments flanking the target sequence.