The objective of this project is the study of the expression of green fluorescent protein cdna in escherichia coli hb101 using puc 18 vector. Recombinant DNA technology is the isolation of a specific DNA segment and its insertion into another DNA molecule at a desired position. The product thus obtained is termed as recombinant DNA and this technique is also known as Genetic Engineering.
Genetic material stores the genetic information of an organic life form. The genetic material for all currently known living organisms is De-oxy Ribo Nucleic Acid (DNA); except some viruses which have Ribo Nucleic Acid (RNA).Genetic recombination involves the movement of genes or gene marker between two distinct chromosomes derived from two sources. Joshua Lederberg and Edward Tatum discovered that transfer of genetic information could occur between two bacterial cells.
Bacterial transformation refers to a genetic change brought about by picking up naked strands of DNA and expressing it for nutrition, repair and diversity. Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA).The effect was first demonstrated in 1928 by Frederick Griffith, an English bacteriologist while searching for a vaccine against pneumonia. Natural transformation has been observed in some gram negative bacteria. Artificial transformation has been demonstrated in a number of bacterial species, most notably in E.coli, where it is used routinely for cloning DNA. E.coli cells are made competent for transformation.
Competence refers to the state in which bacteria of being able to take up exogenous DNA from the environment. Competence is distinguished into natural competence, a genetically specified ability of bacteria that is thought to occur under natural conditions as well as in the laboratory and Induced or artificial competence, arises when cells in laboratory culture are treated to make them transiently permeable to DNA.
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes (BAC, YAC). The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell.
pLITMUS28i is an extensive set of restriction sites in polylinker, many with unique 4-base overhangs having blue or white section. They oppose T7promoters for making RNA transcripts in either direction or double-stranded RNA. pLITMUS 28i is a multi-purpose cloning in vitro transcription phagemid vector. The molecule is a small, double-stranded circle 2,823 base pairs in length (molecular weight =1.8 x 106 Daltons) pLITMUS 28i is isolated from E.coli ER2738 by a standard plasmid purification procedure.
Green Fluorescent Protein (GFP)
Proteomics is the study of gene products encoded by a genome, including a list of genes that are expressed, the time of expression, the type and extent of a post - translational product of gene, the function of the encoded protein and its location in various cellular components. Of this, the most recent addition to the growing family is Green Fluorescent Protein (GFP).
Green fluorescent protein, GFP is a spontaneously fluorescing protein isolated from coelentrates, such as the jelly fish, Aequorea Victoria . GFP is a bioluminescent marker that causes cells to emit bright green fluorescence when exposed to blue or UV light. Fluorescent GFP has been expressed in bacteria, yeast, slime mold, plants, drosophila, zebra fish, and in mammalian cells. GFP can function as a protein tag.
Structure of GFP:
The Green fluorescent protein is comprised of 238 amino acids (26.9 KDa), from the jelly fish, Aequorea Victoria that fluoresces green when exposed to blue light. The wild type of GFP is fluorescent but has several drawbacks including dual peaked excitation spectra, poor photo stability and poor folding at 370C.
The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 Å by multi wavelength anomalous dispersion (MAD) phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of -sheet with an -helix inside and short helical segments on the ends of the cylinder. GFP has a typical beta barrel structure, consisting of one β-sheet with alpha helix(s) containing the chromophore running through the center. Inward-facing side chains of the barrel induce specific cyclization reactions in the tri-peptide Ser65–Tyr66–Gly67 that lead to chromophore formation. This process of post-translational modification is referred to as maturation. The hydrogen-bonding network and electron-stacking interactions with these side chains influence the color of wt GFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water.
Chromophore formation occurs in discrete steps with distinct emission and excitation properties. Its wild type (wt) absorbance or excitation peak is at 395nm with a minor peak at 47 with extinction coefficients of roughly 30,000 and 7,000 m-1cm-1, respectively. The emission peak is at 508nm. Interestingly, excitation at 395nm leads to decrease overtime 395nm excitation peaks and a reciprocal increase in the 475nm excitation band. This presumed photoisomerization effect is especially evident with irradiation of GFP by UV light.
Advantages of GFP:
The advantage of GFP over other markers is the fact that it is not cytotoxic. It does not interfere with normal cellular activity and it is stable even under harsh conditions. Over the last 5 years, this remarkable molecular marker has emerged as one of the most versatile tools in molecular and cellular biology and is being used to investigate an increasing variety of biological processes in yeast, bacteria, animal and plants. The enormous flexibility as an invasive protein marker in living cells allows for numerous other applications such as cell linage tracer, reporter of gene expression and as a potential measure of protein - protein interactions.
GFP as an Active Indicator
The rigid shell in GFP surrounding the chromophore enables it to be fluorescent and protects it from photo bleaching but also hinders environmental sensitivity. Nevertheless, GFPs that act as indicators of their environment have been created by combinations of random and directed mutagenesis. The pH sensitivity of certain mutants and their potential application to measure organellar pH has already been mentioned. It is possible to engineer phosphorylation sites into GFP such that phosphorylation produces major changes fluorescence under defined conditions . The engineered fusion of GFP within the Shaker potassium channel is the first genetically encoded optical sensor of membrane potential .
Depolarization causes at most a 5% decrease in fluorescence with a time constant of approximately 85 ms, but both the amplitude and speed may well improve in future versions. But the most general way to make biochemically sensitive GFPs is to exploit fluorescence resonance energy transfer (FRET) between GFPs of different colour. FRET is a quantum-mechanical phenomenon that occurs when two fluorophores are in molecular proximity and the emission spectrum of one fluorophore, the donor overlaps the excitation spectrum of the second fluorophores the acceptor. Under these conditions, excitation of the donor can produce emission from the acceptor at the expense of the emission from the donor that would normally occur in the absence of the acceptor. Any biochemical signal that changes the distance between the fluorophores or relative orientation of their transition dipoles will modulate the efficiency of FRET . Because FRET is a through-space effect, it is not necessary to perturb either GFP alone but rather only the linkage or spatial relationship between them.
The potential utility of FRET between GFPs was the main motivation for the development most of the mutations. The change in ratio of acceptor to donor emissions is nearly ideal for cellular imaging and flow cytometry because the two emissions can be obtained simultaneously and their ratio cancels out variations in the absolute concentration of the GFPs, thickness of the excitation source, and the absolute efficiency of detection. Because the sample need be excited only one wavelength, which should preferentially excite the donor, FRET is ideal for laser-scanning confocal microscopy and FACS. FRET also causes changes in donor fluorescence lifetime and bleaching rate, but detection of those signals either requires much more sophisticated instrumentation or is destructive