A mixture is a combination of two or more materials which can be separated. When forming a mixture, there is no chemical reaction that takes place and so no new substance is formed. When the constituents of a mixture are combined they can be physically separated (Tillery, Enger & Ross, 2008). A good example is a mixture of sand and iron. When the two elements are mixed they dont result to a new substance, but instead they form a mixture which can later be separated by use of a magnet. The magnet will pick the iron leaving behind the sand. On the other hand, a compound is formed as a result of bonding between various elements. These elements are in a fixed ratio. When constitutes of a compound are chemically combined they result into a new substance. The constituents of a compound are normally divided into their core elements by chemical means. This chemical separation method involves several procedures.
Difference between a Compound and an Element
A combination of various atoms from different substances results to the formation of a compound. For example, Carbon dioxide consists of Carbon ions and Oxygen ions which are held together by electrical attraction. In contrast, an element is made up of same type of atom (Tillery, Enger & Ross, 2008). For example, Oxygen has one molecule so it exits independently. A compound can be broken down into simpler substances through chemical reactions while elements cannot be broken down thus one can be able to identify whether a substance is a compound or an element through chemical reactions. For instance, Carbon monoxide, which is a compound, can be broken down through a chemical reaction to form Carbon and Oxygen atoms while Oxygen, which is an element, cannot be broken into simpler substances. Also, one can identify whether a substance is a compound or an element by looking at the periodic table. In the periodic table, elements have only one capital letter for example, Calcium which is written as Ca. A substance can be identified as a compound or an element by looking at the equation. If the equation has more than one capital letters, then it is a compound, but if there is only one capital letter, it is an element (Tillery, Enger & Ross, 2008).
Difference between Ionic and Covalent Bond
Ionic bond is a bond formed between a non metal and a metal while a covalent bond is a bond formed between two non metals. Ionic bond is formed when the sharing of electron is imbalanced thus the electron from atom X is completely lost to atom Y whereas the covalent bond is formed when the two atoms are able to share the electrons equally. Ionic bonding involves electrostatic attraction between oppositely charged ions in a chemical compound while covalent bond is characterized by the sharing of pairs of electrons between atoms (Stoker, 2012). Covalent bond has a definite shape while ionic bond had no definite shape.
According to Stoker (2012), Ionic bonds have higher melting and boiling points compared to covalent bonds this is because in ionic bonds more energy is required to break all the ionic bonds between the atoms for it to melt while in covalent bonds only little amount of energy is required to break the bonds thus a low melting point and boiling point. For example, when you want to break the bond between Sodium Chloride which has an ionic bond it means that you have to use a lot of energy to break the same bonds to melt Sodium Chloride and the breaking of all bonds will boil the Sodium Chloride. Consequently, ionic compounds have high melting points and boiling points. The electronegativity of atoms results the formation of the covalent bonds. Electronegativity entails atoms ability to attract electrons. Formation of covalent bond is as a result atoms ability to attract electrons. If the power between the two atoms is the same they share the electrons and form a covalent bond, but if one atoms power exceeds the other, then one loses the electron or electrons to the other forming an ionic bond (Stoker, 2012). Compounds with ionic bonds are solid at room temperature while those with covalent bond are in liquid or gaseous form at room temperature.
Formation of Ionic Compounds
When metals from the left side of the periodic table reacts with the nonmetals from the right side of the periodic table an ionic compound is formed because the metal and the nonmetal will contribute electrons to form a pair or pairs of electrons which will not be shared by the two atoms rather it will go to the stronger atom. This is a result of one atom having a strong affinity to attract electrons than its counterpart electrons (Zumdahl, 2009). The nonmetals on the right side of the periodic table have seven valence electrons on their outer orbit while the metals on the left side of the periodic table have one valence electron in their outer orbit thus; the nonmetals on the right side of the periodic table have high electron- negativity. This means that these nonmetals have more power to attract electrons to them. Therefore, the nonmetals on the right side of the periodic table can easily attract the one valence electron from the metals on the left side of the periodic table. At the end, they form an ionic compound and share each others electrons (Tillery, Enger & Ross, 2008). Examples of such ionic compounds are Sodium Chloride (NaCl) and Magnesium Chloride (Mg2Cl3).
Formation of Covalent Bonds
Most of nonmetals on the right side of periodic table have seven valence electrons on their outer orbit (Pace, 2001). Covalent bond is formed when nonmetals from the right side of the periodic table bond with each other because those metals have seven valence electrons on their outer obit thus they just need one electron to be stable. Each atom shares one of its outermost electron thus two electrons are shared between the atoms. In this case, there is attraction between the positively charged atom and shared electrons.
The presence of shared electrons at the edges of two atoms results to the formation of a covalent bond. In this type of bonding none of the atoms loses or gains electrons. Compounds with covalent bonds include Hydrogen Chloride (HCl) and Methane (CH4). Carbon dioxide has a covalent bond between Hydrogen ion and Chlorine ion while Methane has a covalent bond between Carbon ion and Hydrogen ion. The covalent bond formed between these atoms is easy to break since distinct molecules are formed. Thus, this makes the melting and boiling point of covalent bonded compounds low.
References
Pace, N. R. (2001). The universal nature of biochemistry [Special feature]. Proceedings of the National Academic of Sciences, 98, 805 808.
Stoker, H. S. (2012). General, Organic, and Biological Chemistry. New York: Cengage Learning.
Tillery, B. W., Enger, E. E., & Ross, F. C. (2008). SCI110: Integrated science. New York, NY: McGraw-Hill.
Zumdahl, S. S. (2009). Chemical Principles. New York: Cengage Learning.
The world is vastly growing into an incorporated global economy driven by technological astuteness, innovation, the urge for entrepreneurship, and an education whose foundation lies in both natural and social sciences.
There is no doubt that with the rising social problems, people have resorted to research and education in order to solve problems. In various countries, the emphasis on science education is enormous—and scientist are busy working day and night to advance their countries technologically, lest they become doomed in the competitive global age.
For instance, in the study of life sciences, the amalgamation of various academic disciplines only serves to enhance science education. Unlike in the past, people have seen the importance of studying life sciences, and with the support from private and public sectors, the number of students enrolling in life science courses is on the rise.
Life sciences comprises of many disciplines of study, some of them interrelated. In fact, some disciplines in biology, for example, molecular biology, draw most of its dogmas from cell biology. Additionally, the discipline of biotechnology draws many of its dogmas from biochemistry. This paper will discuss the central dogmas of biochemistry and their impacts in biotechnology. It will also explain how the changes in biochemistry affect researches and discoveries in biotechnology (Ninfa, Ballou & Marilee, 2010, pp. 7-9).
To start with, biochemistry is a discipline of life science that mainly deals the chemistry of life. In biochemistry, people learn about the physical and chemical characteristics of molecules. It is important to note that molecules are the basis of living organisms. In other words, biochemistry mainly dwells on the chemical reactions happening in the cells and tissues of living organisms, and the interaction of the chemical compositions of body tissues and cells.
Thus, this makes biochemistry different from the normal chemistry courses. Biochemistry is also different from other disciplines of biology simply because it focuses on the understanding of molecular and atomic minutiae of the cells and tissues of living organisms. In biochemistry, students and researchers also study the chemical bonds within the cells and tissues of living organisms as well as the enzymatic reactions. These are some of the basic dogmas of biochemistry (Hunter, 2000, pp. 7-14).
On the other hand, we look at how the dogmas of biochemistry affect those of the discipline of biotechnology. Biotechnology is the study of various modalities of manipulating chemical substances biologically in order to come up with medicinal products, especially drugs. Clearly, we can see that biotechnology applies the first principles and dogmas of biochemistry in order to come up with solutions to the problems affecting the biochemical composition of living organisms.
We know that the cells of living organisms have genes. Now, having known the chemical composition of cells through biochemistry, biotechnologists use the information to develop genetically engineered species. Additionally, the techniques and dogmas of biochemistry enable the transfer of genes from one species to another—the case in biotechnology (Thieman & Palladino, 2008, pp. 3-7).
There is no doubt that both biochemistry and biotechnology are indispensable disciplines in life science. However, the former is an opulence of the later. In fact, the dogmas and techniques of biochemistry are the trademarks of biochemistry. This means that research discoveries in biochemistry impacts heavily those in biochemistry.
For example, research in biochemistry lead to the development of new biological processes. On the other hand, biotechnologists use these biological processes to come up with new biological products. Additionally, the biochemical study of insulin molecules forms the basis in which biotechnologists will research the corresponding medicines by using biochemical properties (Ninfa, Ballou & Marilee, 2010, pp. 91-97).
Reference List
Hunter, K. (2000). Vital Forces: The Discovery of the Molecular Basis of Life. San Diego: Academic Press.
Ninfa, A., Ballou, D. & Marilee, B. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. (2nd ed.). New York: John Wiley & sons.
Thieman, W. & Palladino, A. (2008). Introduction to Biotechnology. San Francisco: Pearson/Benjamin Cummings.
The obituary article discusses the life and scientific achievements of Michael Smith who lived from 1932 to 2000. Smith grew up in Blackpool, England. In his lifetime he made major contributions to science and earned himself many awards. He is credited for developing ‘site-specific’ mutagenesis. Before he began his work on nucleotide chemistry, Smith graduated from the University of Manchester and became a post Doctoral fellow. This helped him to discover how to change a genetic base. Because of his dedication to science, he received the Nobel Peace Prize. His efforts greatly improved research in Canada. In the late 1980s, he helped found the Biotechnology Laboratory located at the University of British Columbia. He also founded a Center of Excellence in Protein Engineering in Canada. At 64 years of age, Smith worked on genomic, and DNA sequencing at the University of Washington. Since he was a firm believer in genomic research, he accepted the appointment by Victor Ling to become the director of the newly founded Genome Sequence Center. This was at the British Columbia Cancer Agency. Apart from science, people also recognized him for being generous, friendly to family, and zealous for life.
One can see that this article is very resourceful, informative, and inspirational having highlighted the major landmarks in Smith’s intellectual biography. Evidently, the ideas posted are well-structured. This provides readers with constructive insights about the author. The fact that Smith was passionate about science and equally devoted much of his time to society and his country of residence is critical. Most ideologies presented in the article are very enlightening especially in the areas of gene sequencing, other scientific discoveries, and his relationship with colleagues.
Works Cited
Hayden, Michael, and Victor Ling. “Michael Smith (1932–2000).” Nature, vol. 408, 2000, p. 786.
In chemistry, many branches, approaches, and techniques can be applied to clarify the condition of matters or explain the reaction under specific conditions. Spectrophotometry is one of such methods to be applied for measuring the transmittance and absorption properties of chemical substances. Color is everywhere in the human world, and it is possible to use light that passes through solutions and examine an electromagnetic spectrum in wavelengths (Drbodwin, 2010). Spectrophotometry helps conduct qualitative and quantitative analyses in different samples without direct contact. In a biochemical setting, the Beer-Lambert Law is used to determine the concentration of various compounds like nucleic acids, deoxyribonucleic acids, ribonucleic acids, and proteins. The work of a spectrophotometer is based on this law to calculate the amount of light, its absorption, and concentration. Many academic facilities and scientists rely on spectrophotometry results as a simple technique to measure light and understand how color may behave.
Spectrophotometric analysis plays an important role in chemistry, biology, physics, and other natural sciences to learn how the biomolecule concentration of solutions can be determined. In the middle of the 19th century, the final version of Beer’s law was introduced to prove the relationship between transmittance and absorption of a solution. The law states that the way of how a substance absorbs light is proportional to its sample concentration amount; thus, there is a linear relationship between the elements (Khan Academy, 2010). The Beer-Lambert Law introduces the equation:
A = ƐCL, where A is absorbance, Ɛ is the molar extinction coefficient, C is the molar concentration, and L is an optical path length (Drbodwin, 2010). The transmittance, in its turn, is the ratio between the light amount passing through and the incident light. The intensity of light depends on two factors – the level of concentration and the level of absorption. Many teachers use the same example to describe spectrophotometry: the beakers of different sizes (which affects the absorption) and different concentration levels. There is a simple rule in this experiment, which proves that lower concentration leads to higher intensity and vice versa. The spectrophotometer consists of two devices: a spectrometer to analyze the wavelength of light and a photometer to detect the number of absorbed photons.
The biochemical context implies the possibility of using spectrophotometers in many areas of science, including pharmacology, the analysis of water conditions, and laboratory work. Spectrophotometry is necessary to demonstrate how non-destructive methods help check the condition of the water, its quality, and clarity. The presence of heavy metals may challenge people who use this water for drinking or cooking, and it is important to validate the purity of the substance. Chemical scientists may use the same method during the production of pharmaceuticals/ Spectrophotometry allows comparing and measuring samples to understand their compounds and reveal mistakes, if any.
In general, spectrophotometry is commonly used in different natural sciences, including chemistry, biology, biochemistry, and physics. When it is necessary to measure chemical substances or determine reactions, spectrophotometers are offered to analyze the intensity of light absorbed after passing a solution with a certain level of chemical substances (concentration). There are many elements that remain invisible to the human eye, and the worth of such methods as spectrophotometry is a chance to learn better the world around and understand the changes that seem to be unreasonable or poorly investigated.
Protein translocation is the process in which fully synthesized proteins are transported to the target organelle within the cell. This occurs via the heteromeric conduits of the Endoplasmic reticulum. Protein translocation is significant because it enhances the transportation of specific proteins to target organelles. Types of protein translocation processes include cotranslational translocation in addition to posttranslational translocation. This process takes place in different ways depending type of organism that is eukaryotes and prokaryotes as well as the type of proteins being translocated (Halic, 2005).
Types of proteins
There are two types of proteins; this includes secretory proteins as well as membrane proteins. Each type of protein has a different targeting signal, for example, cleavable signals target secretory proteins and this causes the proteins to cross the membrane completely. On the other hand, non-cleaved signals target membrane proteins. Non-cleaved signals are also known as transmembrane (TM) segments. The eventual result of this is the integration of TM segments within the lipid bilayer (Halic, 2005).
Heteromeric channels
The two types of proteins use protein-conducting channels in order for translocation to take place. The occurrence of this channel occurs due to the existence of a preserved heteromeric membrane protein complex. In prokaryotes, this complex is referred to as secY while in eukaryotes it is referred to as sec61. Components of this complex include three subunits namely a, g as well as b-subunits. Each type of subunit has a unique spanning pattern. For example, a-subunit spins within the complex ten times while both g and b-subunits spin within the complex once (Irihmovitch, 2003).
The heteromeric channel has a hydrophilic interior as well as a flexible pore. This allows the passage of large chemicals linked to amino acid side chains. Despite this flexibility, this path remains a barrier as the translocation process takes place thus preventing the passage of ions as well as miniature molecules. Another property heteromeric channel is its ability to open laterally. This allows lateral movement of transmembrane proteins from the hydrophilic environment to the outer hydrophobic layer of the lipid bilayer. The capability of the heteromeric channel to open in two different ways i.e., laterally and across discerns it from other conduits. Heteromeric channels have pores with dimensions ranging from 5–8 A° due to the small size of pores, a mechanism is needed to allow the further opening to facilitate the passing through of large polypeptide chain molecules. This mechanism occurs through lateral dislodgment of helices, which, attaches the pores residues. This property allows the pores to be flexible thus allowing movement of both small and large molecules through (Irihmovitch, 2003).
Forms of protein translocation
Since the sec61 and secY heteromeric channel is a reflexive pore, it allows polypeptide chains to slide back and forth. This necessitates a driving force that allows protein translocation. This is achieved through associations between the channel and various partners. Due to different partners, translocation is divided into three modes namely cotranslational, translocation, posttranslational translocation as well as eubacteria posttranslational translocation (Lurink & Sinning 2004).
Cotranslational translocation
In this form of translocation, the heteromeric channel associates Marjory with the ribosome. This is the most common type of protein translocation and occurs in all classes of organisms as well as cells. Cotranslational translation is responsible for integration between membrane proteins. The first phase of cotranslational translocation is the targeting phase. In this step, a signal recognition particle (SRP) directs a growing ribosome chain into the membrane. The ribosome has an SRP receptor, which also participates in the targeting phase (Halic &Beckmann 2005).
The second phase entails the binding of the ribosome to the heteromeric channel. Upon binding, the growing polypeptide is uncoupled from the ribosome to the heteromeric channel. The movement of the polypeptide chain across the heteromeric channel is facilitated by hydrolysis of GTP. GTP hydrolysis provides the needed energy to drive the proteins or polypeptide chain in a forward direction across the heteromeric channel. In case the ribosome produces a cytosolic sphere of membrane proteins, the protein is translocated laterally along the channel. Eventually, it emerges from heteromeric channel-ribosome junction askew into the cytosol (Mothes et al. 1997).
Posttranslational translocation
This mode of translocation occurs in eukaryotes where polypeptide chains or proteins are translocated after they are fully synthesized. In this mode of translocation, SRP interactions with polypeptide chains during protein synthesis do not exist because proteins translocated by this mode have a minimal hydrophobic signal sequence (Ng et al. 1996).
Posttranslational translocation has been studied and observed in S. cerevisiae and from this observation; the process is similar in higher prokaryotes. In posttranslational translocation, the heteromeric channel partners with luminal BiP protein to produce the driving force. Lumenal BiP proteins belong to Hsp70 class of ATPases. Posttranslational translocation’s driving energy is produced through the ratcheting mechanism. The binding of a polypeptide chain or protein with BiP proteins within the ER lumen prevents the polypeptide chain from sliding backside into the cytosol. The result of this is forward translocation movement (Matlack et al. 1999).
BiP protein is linked with ATP and has a peptide-binding pocket, which, aids in interaction with the Sec63p Lumenal domain. This association causes hydrolysis of ATP leading to the production of energy, which aids in the forward translocation of proteins within the heteromeric channel. Apart from ATP hydrolysis, this association causes closure of peptide–binding pocket thus preventing detachment of proteins being translocated. After the polypeptide has moved a considerable distance, it is received and bounded by another BiP protein molecule and the process goes on and on leading to frontward movement until the polypeptide chain transverses the channel. Release of BiP proteins from the polypeptide-binding pocket occurs after ATP replaces ADP. This allows a second polypeptide chain to be translocated and the process goes on and on. The ratcheting mechanism has some special aspects; this includes the loss of bound cytosolic chaperones by the polypeptide chains before translocation. The essence of this is to facilitate the forward movement of polypeptide chains. Before translocation, fully synthesized proteins are bound to various types of chaperones bind the polypeptide chains specifically at the C-terminus however; upon binding of polypeptide chain with Sec complex via the N-terminal sequences, a chaperone is released (Plath & Rapoport. 2000).
Eubacterial Posttranslational translocation
This type of protein translocation occurs only in eubacteria and is meant for secretory proteins. The heteromeric partner, in this case, is cytosolic ATPase known as SecA. SecA enhances the forward movement of polypeptide chains along the heteromeric channel. Since this type of translocation occurs in prokaryotes such as eubacteria, the heteromeric channel, in this case, is SecY. SecA proteins undergo assenting changes after binding with the polypeptide chain. Some organisms such as Archaea display both cotranslational as well as posttranslational translocation mechanisms. Despite the existence of the two translocation mechanism, it is uncertain how they carry out posttranslational translocation because they lack Sec62/63 as well as SecA complexes (Mori & Ito 2000).
Conclusion
Protein translocation occurs across the endoplasmic reticulum through heteromeric channels to target organelles; protein translocation modes include cotranslational and posttranslational translocation. This depends on the type of protein and type of organism. This process enhances the transportation of specific proteins to the specific organelle. This is enhanced by a specific amino acid sequence, which occurs at the end of each protein. The amino acid sequence acts as a code that directs the protein to a specific organelle. Errors associated with protein translocation lead to the occurrence of genetic diseases, therefore, the discovery of this process has enabled scientists to explain the occurrence of various disorders.
References
Economou A, Wickner W. 1994. SecA promotes preprotein translocation by undergoing ATPdriven cycles of membrane insertion and deinsertion. Cell 78:835–43.
Halic M, Beckmann R. 2005. The signal recognition particle and its interactions during protein targeting. Curr. Opin. Structure. Biol. 15:116–25.
Irihmovitch V, Eichler J. 2003. Post-translational secretion of fusion proteins in the halophilic archaea Haloferax volcanii. J. Biol. Chem. 278:12881–87.
Mori H, Ito K. 2001. The Sec protein-translocation pathway. Trends Microbiol. 9:494–500.
Ng DT, Brown JD, Walter P. 1996. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J. Cell Biol. 134:269–78.
Plath K, Rapoport TA. 2000. Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum. J. Cell Biol. 151:167–78.
In the past few decades, Green Fluorescent Protein (GFP) has gained popularity in the fields of cellular biology, biotechnology and microscopy (Chalfie & Kain 2005). The GFP is made up of a total of 238 amino acid residues. These amino acid residues are responsible for the green fluorescence that is a characteristic of the GFP under UV light. GFP has the capacity to transfer energy and through this process, the protein transduces the blue chemiluminescence of aequorin, another type of protein, into green fluorescent light (Snapp 2009). GFP was first discovered by Shimomura and colleagues in 1962. Since then, GFP has continued to attract significant interest from various scientists. The protein is now among the most exploited and studied proteins in biology and biochemistry (Tsien 1998). Its phenomenal capability to produce a noticeable fluorosphore, coupled with its highly organized structure ensures that GFP remains an ideal protein model for study. With time, more universities and other institutions of higher learning will continue to rely on GFP as a standard teaching tool in the field of molecular biology and biochemistry (Tsien 1998). The current essay is an attempt to examine the applications of Green Fluorescent Protein (GFP) in cell biology, biochemistry, and biotechnology. To do so, the essay shall endeavor to outline the sources of GFP. In addition, its structural features shall also be explored, along with how this has played a role in its usefulness. Moreover, the general strategies that are essential for the application of GFP in cell biology, biochemistry and biology shall also be examined. In addition, the essay shall also attempt to explore two experimental studies where GFP has been applied.
Sources of GFP
GFP was first isolated from the jellyfish Aequoria aequoria by Shimomura and colleagues in 1962. While undertaking the isolation, the authors noted in their study that one of the distinctive properties of the Aequoria aequoria is that it had the ability to emit a greening luminescence. While carrying out the isolation, Shimomura et al (1962) also observed that the aequoria emitted blue luminescence, as opposed to the green luminescence that often characterizes the intact organism. Once the authors had made detailed observation of the green luminescence, the protein was thereafter purified characterized, and assigned the name Green Fluorescent Protein (GFP).
Most bioluminescent coelenterates in the classes of anthrioza and hydrozoa contain high levels of Green Fluorescent Proteins. It is yet to be clarified whether GFPs occur in the phylum ctenophore and among the scyphozoans. Naturally occurring GFPs emit light of between 490 and 520 nm (Misteli & Spector 1997). The hydrozoan jellyfish is the only known naturally occurring GFP molecule and has an excitation maximum in the UV (Ultraviolet) region of 395 nm. Traditionally, GFP is used in reference to the protein that Shimomura et al (1962) first isolated from Aequorea Victoria. However, other marine organisms have also been shown to possess the same green fluorescent proteins. Since the characterization of Aequores GFP, scientists have continued with their work on other types of GFPs, leading to the characterizations of additional GFPs like Renilla. The basic chromophore features of both Aequorea and Renilla GFP are almost identical, the only difference being that Renilla GFP has a much higher extinction coefficient in comparison with that of Aequorea GFP. Moreover, Renilla GFP has been shown to be resistant to PH-induced denaturation and conformational changes (Harpur, Wouters & Bastiaens 2001), not to mention that it also has the tendency to dimerize.
Physical and Spectral Properties of GFP
GFP has a documented absorbance peak at 295 nm. However, the average emission peak of FP is 508 nm. As the excitation continues the 395 nm peak declines with time (Zimmer 2002). GFP typical GFP structure consists of a single β-sheet that is covalently bonded to the HBI chromophore. The β-sheet also runs right through the center of the GFP (Zimmer 2002). HBI is not able to fluorescence when the GFP scaffold is missing. The GFP barrel consists of inward-facing side chains whose role is to induce certain cyclinization reactions. This in turn induces HBI ionization to the chromophore and phenolate in a process referred to as maturation. The interaction of the GFP barrel side chains with hydrogen bonding and electron-stacking has a huge effect on the color and intensity of GFP. In addition, it also affects the derivatives of GFP in the same way. The GFP barrel is normally tightly-packed, effectively excluding solvent molecules. This is important for the protection of the chromophore fluorescence against quenching by water.
Single GFP molecules are reported to display off/on switching and blinking when observed photochemically. Once the GFP has emitted a given number of photons, it tends to switch off to what is known as a dark state, lasting for approximately 5 minutes. It is important to note that the dark state differs from the off-blinking behavior (Dickson et al 1997). According to Garcia-Parajo (2000), blinking is largely photo-induced. Separately, Dickson et al (1997) suggest that a reversible changeover between a dark transition of an unknown identify and the emitting anionic form could be the reason behind this behavior.
Fluorosphore
In GFP, an internal Ser-Tyl-Gly sequence acts as the originator of fluorosphore. As such, the fluorosphore is made up of Ser65-dehydroThy66-Gly67 residues of the GFP protein. The backbone of these three residues is cyclic in nature and it forms an imidazolidone ring (Zimmer 2002). However, the Ser-Tyr-Gly tripeptide does not have fluorescence, and this is indicative of the fact that the GFP fluorescence is very unique. The fluorosphore comes about as a result of an autocatalytic process, meaning that the sequential mechanism does not require any enzymatic reactions or cofactors. Gly 67 and Ser 65 form a rapid cyclization thus initiating an auto-catalytic process (Creemers et al 2000). Next, the Tyr 66 side chain is oxidized leading to the formation of the fluorosphore. This is a thermosensitive reaction. There is a decline in the amount of fluorosphore formed. On the other hand, GFP is largely stable once formed.
GFP structure
A lot of effort has been dedicated to studies involving the chromosphores of GFP-like proteins. These studies are useful in that they have helped to shed light on the chemical structure of chromophores. In addition, the studies have also helped to shed light on how the GFP molecule is formed. One of the defining characteristics of the GFP structure is its cylindrical fold made up of two protomers (Gerdes & Kaether 1998). By studying the crystal structure of GFP, scientists have revealed that the ability of the chromophore to fluorescence is the main reason why GFP has found wide application in various fields.
Figure 1: A solid-state structure of GFP. (Source: Zimmer 2002).
An 11 stranded β-barrel is involved in the formation of GFP. This strand folds into a β-can structure (Zimmer 2002), thereby completely burying the chromophore into the epicenter of the β-can structure. The β-can structure helps to protect the chromophore against the effects of solvent-quenching. Other proteins cannot also reach the chromophore region of the GFP protein since it is folded into the β-can structure. Therefore, the spontaneous formation of chromophore requires a nonenzymatic mechanism. It would also be very hard to truncate GFP to also molecular weight owing to the β-can structure (Shaner, Patterson & Davidson 2007). Nearly the whole of the GFP sequence takes part in the creation of the β-can structure. As such, if any part of the sequence is to be deleted, the entire conformation would be disrupted.
Moreover, multiple covalent interactions help to stabilize the barrel of the GFP.
Owing to this stability, the GFP demonstrates high stability to chemical and thermal denaturation (Shaner et al 2007). The GFP also tends to be quite resistant to proteolysis. Other GFP- like proteins also demonstrates a tendency to oligomerize.
GFP Application in Cell Biology
GFP has found wide application in cell biology as a biological marker following the discovery that its fluorescence does not require a cofactor. In addition, the use of GFP does not change the normal localization or function of the enjoined partner. Also, in order to localize GFP, it is not necessary to undertake substrate entry and fixation permeabilization (Day 1998). Consequently, organelles, cells and proteins that have been marked with GFP can easily be examined in living situations. This breakthrough has enabled biologists to investigate the developmental processes and cellular dynamics in intact cells. Other than the broad impact that GFP technology has had on basic research, the protein has also found use in such areas as assessment of viral vector in human genome therapy (Bastiaens & Pepperkok 2000). GFP is also increasingly being used in monitoring the activity of genetically altered microbes, high throughput drug screening and biological pest control. More importantly, GFP technology has found application in the discovery of drugs.
Some of the properties of GFP that renders it applicable in drug discovery include ease of use, real-time kinetics, and cost savings (Chalfie et al 1994; Chudakov et al 2010). These properties have seen GFP replace other markers like β-galactose and luciferase. GFP fluorescence has also found wide application in determining a significant collection of behaviors and properties. This is mainly the case because the production process of the chromophore of GFP occurs via an internal post-translational autocatalytic cyclization. This cyclization does not need any substrates of cofactors. In addition, the protein’s mobility or activity is rarely affected by the fusion of GFP to a protein (Wouters, Verveer & Bastiaens 2001). Also, GFP is largely nontoxic, not to mention that it is also reported to show resistance to alkaline PHJ, photobleaching, heat, detergents, organic salts, chaotropic salts, and a host of proteases.
Reviews of GFP application in microscopy, biotechnology and cell biology GFP and as a reporter gene have been published. A GFP gene can be used to monitor gene expression by measuring the GFP fluorescence. In this case, GFP fluorescence acts as a direct indicator of the level of gene expression in tissue. Although GFP has been widely used as a reporter gene, on the other hand, the lack of signal amplification means that it has low sensitivity (Zimmer 2002). This is because there is only one chromophore for each GFP, and this tends to limit its use.
GFP has also been used as fusion tags. In this case, the GFP is fused with a cloned gene using standard subcloning methods. The ensuing chimera is expressed in an organism or cell (Bastiaens & Pepperkok 2000). Such GFP fusion tags are very useful in monitoring protein localization, in addition to visualizing dynamic cellular events. Since GFP does not need any substrates or cofactors, this makes it an ideal fluorescence fusion protein marker. The production of the chromophore takes place in vivo, while the ensuing chimera rarely affects the activity or localization of tagged proteins. Consequently, GFP fusion proteins have found wide application in the field of biotechnology.
GFP has found valuable application as a biological marker since its fluorescence does not require any additional cofactors. When the peptide backbone of the molecule crystallizes the fluorosphore is formed (Zimmer 2002). This enables scientists to locate the position of proteins in cells. In addition, Zimmer (2002) reports that the reason why scientists prefer using GFP as a tag is that it does not interfere with the localization of the fusing partner. GFP is also actively used as a cell marker. Other scientists report of its use as an active indicator of calcium sensitivity. Besides, GFP plays an important role in transcription factor dimerization (Bastiaens & Pepperkok 2000), as well as protease action. Recently, biochemical engineers have also discovered that GFP has the potential to quantitatively assess gene expression in various organisms.
Modified forms of GFP have also found use in the making of biosensors. Scientists have managed to introduce the GFP gene into organisms and at the same time, maintain it in their genome through vector injection, bleeding, or cell transformation (Pepperkok et al 1999). GFP is also increasingly being used in FRE (fluorescence resonance energy transfer) applications (Bastiaens & Pepperkok 2000; Wouters, Verveer & Bastiaens 2001). The reason why some of the spectral variants of GFP are seldom utilized as FRET partners is that the donor-acceptor emission spectra are not entirely separated, while the chromophore is located at the very core of the GFP. Also, some of the spectral variants of GFP tend to fluorescence at comparatively low intensities. FRET was first utilized as a calcium probe (Miyawaki et al 1999). In this case, FRET has found use in the determination of calcium concentrations. Day (1998) also reports that FRET has been quite valuable in studies involving protein-protein interactions. Separately, Harper (2001) has published a paper on FRET imaging technique capable of getting around the need for two spectrally unique FRET partners. In this study, Harper (2001) reports on the use of fluorescent lifetime imaging microscopy (FLIP) as a technique for evaluating the fluorescence lifetime released by an EGFP/EYFP pair that was earlier unusable and spectrally similar for FRET. Creemers et al (2000) also report that GFP has found application in high-resolution, low-temperature spectroscopy.
Experimental studies involving GFP
Ohashi et al (2007) conducted an experimental study of GFP-based fluorescence resonance energy transfer (FRET). In their experiment, the authors inserted intrinsically or folded unstructured proteins between Ypet and Cypet. In their experimental study, Ohashi et al (2007) also revealed that enhanced dimerization of GFP molecules led to improved FRET signal. In addition, the researchers recorded a moderate FRET signal with single a fibronectin insert, but no FRET signal was recorded with the double fibronectin. Based on their experimental study, the authors concluded that GFP-based FRET might prove crucial for examining intrinsically unstructured proteins.
Another study carried out by Pepperkok et al (1999) sought to detect multiple green fluorescent proteins using fluorescence microscopy. The multiple gene fluorescence protein was being detected in live cells. The authors used fluorescence lifetime imaging microscopy to demonstrate how GFP variants had managed to yield discernible fluorescence lifetime. Those co-expressed GFP variants that demonstrated similar fluorescence intensities had to be separated using lifetime images. This enabled the researchers to open up extra spectroscopic dimensions thereby generating wavelengths that facilitated the selection of novel GFP variants.
Conclusion
Green Fluorescent Protein (GFP) is increasingly becoming popular in developmental and cellular biology, biotechnology, and microscopy because it has a phenomenal ability to produce noticeable fluorosphore. Shimomura et al (1962) first isolated GFP in 1962. Most bioluminescent coelenterates contain high levels of GFP. However, the hydrozoan jellyfish is the only known naturally occurring GFP molecule, while the Aequoria GFP genes are the only GFP genes to have been cloned thus far. GFP has a maximum absorbance peak at 295 nm. Photochemical observation of single GFP reveals an on/off switching and blinking display. The Ser-Tyl-Gly sequence acts as the originator of fluorosphore, which comes about owing to an auto-catalytic and thermosensitive reaction process. The basic structure of GFP entails an 11 stranded β-barrel that folds into the β-can structure. Multiple covalent interactions ensure that the barrel remains stable. Over the past few decades, GFP has found wide application in the fields of cell biology, microscopy, and biotechnology, among other fields. GFP is also increasingly being used in the discovery of new drugs owing to its ease of use and cost savings. As a result, it has now replaced other biological markets like β-galactosidase and luciferase. In the fields of microscopy, biotechnology and cell biology, GFP is being used as a reporter gene, as fusion tags, and as biosensors.
Reference List
Bastiaens, PIH & Pepperkok, R 2000, ‘Observing proteins in their natural habitat: the living cell’,Trends Biochem. Sci., Vol. 25, pp. 631-637.
Chalfie, M & Kain, SR 2005, Methods of Biochemical Analysis, Green Fluorescent Protein: Properties, Applications and Protocols, John Wiley & Sons, New York.
Chalfie M, Tu Y, Euskirchen G, Ward WW & Prasher DC 1994,’ Green fluorescent protein as a marker for gene expression’, Science, Vol. 263, pp. 802–805.
Chudakov DM, Matz MV, Lukyanov S & Lukyanov KA 2010,’ Fluorescent proteins and their applications in imaging living cells and tissues’, Physiol Rev., Vol. 90, pp. 1103–1163.
Creemers, TMH, Lock, AJ, Subramaniam, V, Jovin, TM & Volker, S 2000,’ Photophysics and optical switching in green fluorescent protein mutants. Proceedings of the National Academy of Sciences, Vol. 97, pp. 2974-2978.
Day, RN 1998,’ Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol Endocrinol., Vol. 12 no. 9, pp. 1410-9.
Dickson RM, Cubitt AB, Tsien RY & Moerner WE 1997,’ On/off blinking and switching behaviour of single molecules of green fluorescent protein’, Nature, Vol. 24 no. 388, pp. 355-358.
Garcia-Parajo MF, Segers-Nolten GM, Veerman JA, Greve J & van Hulst NF 2000,’ Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules’, Proc Natl Acad Sci., Vol. 97 no. 13, pp. 7237–7242.
Gerdes, HH & Kaether, C 1996,’ Green fluorescent protein: applications in cell biology’, FEBS Lett., Vol 389 no. 1, pp. 44-7.
Harpur, AG, Wouters, FS & Bastiaens, PI 2001,’ FRET between spectrally similar GFP molecules in single cells’, H. Nat. Biotechnol., Vol. 19, pp. 167-169.
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Miyawaki, A, Griesbeck, O, Heim, R & Tsien, R 1999,’Calcium as a Cellular Regulator’, Proc. Nat. Acad. Science U.S.A., Vol. 96, pp. 2135-2140.
Ohashi, T, Galiacy, SD, Briscoe, G & Erichson, HP 2007,’An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins’, Protein Science, Vol 16, pp. 1429–1438.
Pepperkok, R, Squire, A, Geley, S & Bastiaens, PIH 1999,’Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy’, Current Biology, Vol. 9, No. 5, pp. 269-274.
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Shimomura, O, Johnson, FH & Saiga, Y 1962,’ Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan’, Aequorea. J. Cell. Comp. Physiol., Vol. 59, pp. 223–239.
Tsien, RY 1998,’The Green Fluorescent Protein’, Annual Review of Biochemistry, Vol. 67, pp. 509-544.
The biochemical cycles known by another name the nutrient cycles involve a process by which there is a noticeable change in an element’s physical or chemical structure as it moves through the different systems of the earth.
These systems include the atmosphere, lithosphere, biosphere and the hydrosphere. It is important for these cycles to occur as they help in replenishing elements that may not be freshly produced hence making them available at all times.
Most of these elements that undergo recycling are the very vital elements or nutrients required by all,l living things to survive, for example, water, carbon, nitrogen just to mention, but a few.
A nutrient is, therefore, a substance that is required by living things for their survival (Bolin, 2001, p.1). There are macronutrients that are required in larger quantities in the body, and these include carbon, phosphorous, carbon, oxygen, nitrogen and hydrogen.
As for the micronutrients, they are required in small amounts but are also essential. Boron, copper, and molybdenum fall in this category.
Major Biogeochemical Cycles
The major biochemical cycles that occur on earth are the carbon, water, nitrogen, hydrogen, and the sulfur among others. The water cycle is very important to all living things since it is an essential nutrient for growth and life.
It is also known as the hydrological cycle and mainly involves evaporation of water from the earth’s surface to the atmosphere where it condenses then falls in the form of precipitation collecting in the sea, lakes, rivers or sinks down to form the groundwater.
The process is thus repeated forming a cycle. The nitrogen cycle is another major cycle which occurs by nitrogen from the atmosphere being fixed into the soil through a process known as nitrogen fixation mostly carried out by organisms such as algae and bacteria.
Carbon is also an important element for the living organisms; hence the carbon cycle which is a gaseous cycle takes place to recycle the carbon and make it available for the living organisms.
Carbon is found in living or organisms that have died recently, in the atmosphere, in water bodies and dead organisms in the form of calcium carbonate. There is also the sulfur cycle, though required in limited amounts because of safety sulfur is also very important.
It involves the gaseous sulfur in the form of sulfur dioxide or particulate sulfur found in the air. The other major biochemical cycle is the phosphorous cycle; this is a sedimentary cycle providing energy to living organisms.
Micronutrients though required in minimal amounts; they play a very important role in the global cycles as some activities to support the major cycles while others accelerate the major cycles (Bolin, 2001, p.1).
Global cycles are those that act to support the earth’s system. An example of a global cycle is the carbon cycle. It occurs when plants absorb carbon dioxide from the atmosphere and use it to manufacture food.
The animals feed on the plants, and upon their death, the animals decay hence deposited to form coal or petroleum. On the other hand, the sea creatures get the dissolved carbon dioxide from the atmosphere which has combined with the water.
The shells of the dead creatures form at the base of the water body forming limestone. The cycle continues; hence carbon is recycled.
The impact of the carbon cycle is that there may be excess carbon that may be produced in the atmosphere and hence being poisonous. Diagram showing the carbon cycle
Fig. (Bolin, 2001, p.1)
Conclusion
All biochemical cycles however major or minor the nutrient involved is are important to the environment. This is because they help in replenishing the nutrients making them available to living things at all times.
Reference List
Bolin, P. (2001). The Major Biogeochemical Cycles and Their Interactions. Web.
It is known that naturally contained in water layers organic substances are subject to bacterial destruction. Aerobic microorganisms use molecular dissolved oxygen for their own metabolic processes, and as a product of synthesis, emit carbon dioxide (CO2). In other words, dissolved oxygen actively diverges into oxidation, which reduces its concentration in water over time. Thus, by measuring the oxygen level in a water sample, it can be determined the activity of bacteria and the concentration of organic matter: the more oxygen is consumed, the more organisms absorb it. (Hang, 2017). A quantitative measure of this fact is biochemical oxygen demand or BOD.
The essence of the method of measuring BOD is to establish the concentration of molecular oxygen in the water sample immediately after the selection and after incubation periods (5, 7, 10, or 20) of the sample (UmweltBundesamt, 2017). The microorganisms that have absorbed oxygen emit carbon dioxide captured by the added amount of potassium hydroxide. This causes changes in pressure, which is recorded by the device.
Procedure
Sample Preparation
Water samples should be brought to the target pH value (6.5-7.5) by adding the required amount of low concentrated (1 M) solutions of sulfuric or hydrochloric acid;
The sample should be homogenized by mixing and filtration, if necessary.
The representative volume of the material is placed in an empty, clean measurement container.
In order to eliminate unwanted nitrification, inhibitor B is placed in the solution, the mass of which is set by the standards depending on the range to be determined.
Next, 3-4 drops of 45% solution of potassium hydroxide are added to the sealing lining, and a magnetic anchor is placed in the sample.
The required temperature of the sample can be set by heating it in a thermostatic cabinet.
Container caps are tightly screwed, it is important to achieve absolute tightness of the system.
Measurements
BOD Direct™ sensors are placed directly on the water sample so as to avoid air ingress or removal. Airtightness is the main measurement process.
Molecular oxygen is measured automatically.
The samples are incubated during a set period. this can be 5, 7. 10, or 20 days. During this period, the bacteria actively consume oxygen.
Once a specific deadline is reached, the measurement is repeated as described in steps 8.
The numerical data are recorded in the table and the total biochemical oxygen consumption is calculated.
A mixture is a combination of two or more materials which can be separated. When forming a mixture, there is no chemical reaction that takes place and so no new substance is formed. When the constituents of a mixture are combined they can be physically separated (Tillery, Enger & Ross, 2008). A good example is a mixture of sand and iron. When the two elements are mixed they don’t result to a new substance, but instead they form a mixture which can later be separated by use of a magnet. The magnet will pick the iron leaving behind the sand. On the other hand, a compound is formed as a result of bonding between various elements. These elements are in a fixed ratio. When constitutes of a compound are chemically combined they result into a new substance. The constituents of a compound are normally divided into their core elements by chemical means. This chemical separation method involves several procedures.
Difference between a Compound and an Element
A combination of various atoms from different substances results to the formation of a compound. For example, Carbon dioxide consists of Carbon ions and Oxygen ions which are held together by electrical attraction. In contrast, an element is made up of same type of atom (Tillery, Enger & Ross, 2008). For example, Oxygen has one molecule so it exits independently. A compound can be broken down into simpler substances through chemical reactions while elements cannot be broken down thus one can be able to identify whether a substance is a compound or an element through chemical reactions. For instance, Carbon monoxide, which is a compound, can be broken down through a chemical reaction to form Carbon and Oxygen atoms while Oxygen, which is an element, cannot be broken into simpler substances. Also, one can identify whether a substance is a compound or an element by looking at the periodic table. In the periodic table, elements have only one capital letter for example, Calcium which is written as Ca. A substance can be identified as a compound or an element by looking at the equation. If the equation has more than one capital letters, then it is a compound, but if there is only one capital letter, it is an element (Tillery, Enger & Ross, 2008).
Difference between Ionic and Covalent Bond
Ionic bond is a bond formed between a non metal and a metal while a covalent bond is a bond formed between two non metals. Ionic bond is formed when the sharing of electron is imbalanced thus the electron from atom X is completely lost to atom Y whereas the covalent bond is formed when the two atoms are able to share the electrons equally. Ionic bonding involves electrostatic attraction between oppositely charged ions in a chemical compound while covalent bond is characterized by the sharing of pairs of electrons between atoms (Stoker, 2012). Covalent bond has a definite shape while ionic bond had no definite shape.
According to Stoker (2012), Ionic bonds have higher melting and boiling points compared to covalent bonds this is because in ionic bonds more energy is required to break all the ionic bonds between the atoms for it to melt while in covalent bonds only little amount of energy is required to break the bonds thus a low melting point and boiling point. For example, when you want to break the bond between Sodium Chloride which has an ionic bond it means that you have to use a lot of energy to break the same bonds to melt Sodium Chloride and the breaking of all bonds will boil the Sodium Chloride. Consequently, ionic compounds have high melting points and boiling points. The electronegativity of atoms results the formation of the covalent bonds. Electronegativity entails atoms ability to attract electrons. Formation of covalent bond is as a result atoms ability to attract electrons. If the power between the two atoms is the same they share the electrons and form a covalent bond, but if one atom’s power exceeds the other, then one loses the electron or electrons to the other forming an ionic bond (Stoker, 2012). Compounds with ionic bonds are solid at room temperature while those with covalent bond are in liquid or gaseous form at room temperature.
Formation of Ionic Compounds
When metals from the left side of the periodic table reacts with the nonmetals from the right side of the periodic table an ionic compound is formed because the metal and the nonmetal will contribute electrons to form a pair or pairs of electrons which will not be shared by the two atoms rather it will go to the stronger atom. This is a result of one atom having a strong affinity to attract electrons than its counterpart electrons (Zumdahl, 2009). The nonmetals on the right side of the periodic table have seven valence electrons on their outer orbit while the metals on the left side of the periodic table have one valence electron in their outer orbit thus; the nonmetals on the right side of the periodic table have high electron- negativity. This means that these nonmetals have more power to attract electrons to them. Therefore, the nonmetals on the right side of the periodic table can easily attract the one valence electron from the metals on the left side of the periodic table. At the end, they form an ionic compound and share each other’s electrons (Tillery, Enger & Ross, 2008). Examples of such ionic compounds are Sodium Chloride (NaCl) and Magnesium Chloride (Mg2Cl3).
Formation of Covalent Bonds
Most of nonmetals on the right side of periodic table have seven valence electrons on their outer orbit (Pace, 2001). Covalent bond is formed when nonmetals from the right side of the periodic table bond with each other because those metals have seven valence electrons on their outer obit thus they just need one electron to be stable. Each atom shares one of its outermost electron thus two electrons are shared between the atoms. In this case, there is attraction between the positively charged atom and shared electrons.
The presence of shared electrons at the edges of two atoms results to the formation of a covalent bond. In this type of bonding none of the atoms loses or gains electrons. Compounds with covalent bonds include Hydrogen Chloride (HCl) and Methane (CH4). Carbon dioxide has a covalent bond between Hydrogen ion and Chlorine ion while Methane has a covalent bond between Carbon ion and Hydrogen ion. The covalent bond formed between these atoms is easy to break since distinct molecules are formed. Thus, this makes the melting and boiling point of covalent bonded compounds low.
References
Pace, N. R. (2001). The universal nature of biochemistry [Special feature]. Proceedings of the National Academic of Sciences, 98, 805 – 808.
Stoker, H. S. (2012). General, Organic, and Biological Chemistry. New York: Cengage Learning.
Tillery, B. W., Enger, E. E., & Ross, F. C. (2008). SCI110: Integrated science. New York, NY: McGraw-Hill.
Zumdahl, S. S. (2009). Chemical Principles. New York: Cengage Learning.
The teenage years are an important period in a person’s life. It is the part of childhood when people learn the most about themselves. It is also the time when their brains and bodies undergo serious chemical and biological changes. These aspects make the teenage years universally understood as difficult and volatile; however, these changes often come as a surprise to both the parents and the teenagers.
The three presented videos clearly show that it is normal for a teenager to strive towards independence or even danger. Siegel explains it as a biological factor, nature’s way of making teenagers explore and learn more about the world (Siegel 3:30 – 4:00). Each of the videos used data collected by MRI to provide insight into how an adolescent brain works. More specifically, the MRI results point out the growth spurt that occurs in the prefrontal cortex before puberty and then a decrease in the grey mass, which is called pruning. It was a fascinating aspect to find out that growing up includes not only growing parts of the body or brain, but also discard of certain elements. The main difference between the videos lies in ways that the information is delivered, be it through a report-like presentation, personal interviews, or a speech at a large gathering.
It is most likely that for teenagers of all generations, the strive to be independent and free will be relevant in one shape or form. The adolescence of the past and present undoubtedly share certain unruly features, such as talking back or showing disrespect to authority. However, in the current realities, this aspiration could be decelerated by social media and the Internet. The need to achieve and learn more could be satisfied vicariously through watching videos or reading about people’s experiences in media. In addition, the parental methods have changed since 2002, becoming less strict, which creates less tension between the parents and the children.