1. Cell Transformation:
Cell transformation, or bacterial transformation, is a process of developing and upmost importance in modern molecular biology [6]. With this process, genetically engineered or naturally occurring plasmids may be introduced into bacterial cells [6]. From there the “new DNA” is decoded by the cell and expresses the trait specifically desired by the engineering process [6]. The bacteria are thus used to amplify the products of the decoded plasmid on a large scale [2]. This is based on the function of the plasmid to deliver genetic material vital to the survival of the bacteria [2]. However, it is not the only way in which DNA is transferred to bacterial cells as there is also transfection and conjugation [17]. In transfection small viruses called bacteriophages inject foreign DNA into their hosts [17]. Conjugation involves the mating between two different bacterial cells and the transfer of their DNA to one another [17]. In transformation, however, information written in DNA necessary for the survival of cells in the colony, is passed through the environment to surrounding cells throughout the colony.
The transformation process involves the uptake of foreign DNA by a cell which results in a newly acquired genetic trait that is coded by the foreign DNA – plasmid [6]. This trait is now stable and heritable after transformation is complete [8]. Before they can accept the foreign DNA, however, bacterial cells must first reach a particular physiological state of competency [6]. DNA is an extremely hydrophilic (water loving) molecule and thus will not easily pass through a bacterial cell membrane on its own [2]. Natural genetic competency is the ability of a cell to bind and take up foreign, or exogenous, DNA [14]. Cells in this natural state express specialized proteins that assemble into a DNA-uptake complex [14]. In many organisms the development of competence is a response either to regulated cell-to-cell signaling or in response to nutritional conditions [24]. A cell may be forced into competency by making small holes in the cell membrane by suspending the cells in a solution of concentrated calcium [2]. This process, of exposing cells to solutions which alter the cell membrane enough to allow small molecules to pass, is called chemical transformation [17]. Electroporation is another form of cell transformation not featured in this experiment that involves passing short and high voltage currents through a solution of cells [17]. Chemical transformation creates holes in the cell membrane and thus allows a place for the hydrophilic DNA plasmids to pass.
A plasmid is a small circular piece of genetic information or DNA that encodes a trait important for the growth or survival of the bacteria [2]. It consists of only about 2,000 to 10,000 base pairs [2]. In nature, a plasmid is, in most cases, a gene that encodes a protein making those bacteria resistant to antibiotics, an ability that probably evolved from the growth of bacteria in close proximity to other heterotrophs [2]. While bacteria and their fungi and mold neighbors would compete for the same food source, the fungi and mold would secrete toxins used to kill these bacteria, similar to antibiotics used in medicine [2]. Plasmids are not involved in reproduction and only the chromosome in the cell carries genetic material necessary to carry out binary fission, the primary means of reproduction [4]. Though plasmids are not entirely necessary for survival they do give bacteria a colonial advantage [4], almost like inter-bacteria communication. When plasmids are genetically engineered, scientists also may add genes coding proteins that produce a distinctive color when the plasmid has been accepted and coded by the bacteria [17]. This allows for much easier identification of transformed cells. Much research is being done to determine the exact pathways plasmid DNA follows within the cytoplasm of the prokaryotic cell and then to coding by the prokaryotic cell.
Transformation efficiency is a measure of the amount of cells within the bacterial culture that are able to take up the plasmid DNA and may be determined experimentally [17]. It is defined by the number of transformants per microgram of DNA [6]. For some microbiology experiments, such as cloning and sub-cloning, high transformation efficiency is not critical [17]. For others, like a construction of a genomic library, high transformation efficiency is absolutely critical [17].
Cell transformation has much use to society and science now and in the future. Transformation is used for cloning or moving around of DNA molecules within the strain [17]. Bacteria are transformed to produce many necessities in medicine as well, such as insulin or vaccines [17]. This illustrates the large spectrum of application of transformation as the same species could produce a recombinant protein used in treating disease, like insulin, or one used in preventing it, like a vaccine. In another sector, transformation of bacteria to ensure their survival under certain conditions would be extremely useful in environmental “clean-ups” through bioremediation [17]. The production of small peptides (enzymes or proteins that are coded in genes) without cell transformation is enormously expensive, and much more so for larger peptides [17]. Thus the use of cells to code for large peptides is also economically friendly and favorable.
2. Cell Transformation in E. coli:
Most of the current transformation experiments involve E. coli, though E. coli does not enter a state or competency unless artificially induced [6]. The transformation process featured in this experiment uses the pGAL plasmid. This plasmid contains 6751 base pairs and does not integrate into the bacterial chromosome but rather replicates autonomously [6]. The pGAL plasmid contains the E. coli gene that codes for β-galactosidase, known as lacZ [6]. When β-galactosidase is successfully produced in a cell in the presence of the organic compound X-Gal, as done in this experiment, a distinct blue coloring will be evident and thus a reflection of successful transformants [6]. The primary function, though, of this plasmid is to encode a gene producing an enzyme, β-lactamase, inactivating, and thus making the cell resistant to ampicillin, a modern antibiotic; the rest of the plasmid is used to create a visual on transformation efficiency [6].
Beta-lactamases are the most common source for resistance to β-lactam antibiotics [11]. The β-lactam family of antibiotics, including five smaller families, all contains a characteristic four-membered ß-lactam (azetidin-2-one) ring [7]. The extended family consists of: the penicillins; clavulanic acid; the carbapenums; the norcardicins and monobactums; and the cephalosporins, cephamycins and cephabacins [7]. These antibiotics inhibit the biosynthesis of the bacterial cell wall thus killing the bacteria [7]. The β-lactamases are bacterial enzymes that catalyze the hydrolysis of the characteristic β-lactam ring [7]. Ampicillin is a member of the penicillin family and is used to treat many different types of infections caused by bacteria, such as ear infections, bladder infections, pneumonia, gonorrhea, and E. coli or salmonella infection [18]. As a β-lactam antibiotic, ampicillin functions by preventing bacterial cells from forming proper cell walls. Also as a β-lactam antibiotic, ampicillin is deactivated by β-lactamase by its ability to catalyze the hydrolysis of the β-lactam ring.
The lacZ gene in E. coli is encoded on the lac operon [10], or grouping of genes that encode similar products involved in similar processes under common control [13]. The lac operon is not constitutively expressed, meaning it is always turned “off” rather than turned “on” [10]. Its expression must be regulated because it is products are only needed under certain conditions. E. coli usually grows in a medium based on glucose and therefore feeds on this available glucose, however, if placed in a medium of lactose rather than glucose, the cells would need to metabolize the lactose [10]. The ability to do so is encoded on the lac operon, which is usually suppressed by the presence of glucose and lack of lactose [10]. The lacZ gene in particular encodes for the β-galactosidase enzyme, and in the presence of lactose the quantity of this enzyme rises from none to almost two percent of the weight of the cell [10]. Beta-galactosidase hydrolyzes the breakdown of lactose into glucose and galactose (much like it hydrolyzes the breakdown of X-Gal into its component parts) [10].
Lactose is a sugar molecule composed of two hexagonal rings: each ring is made of five carbon corners and one oxygen corner, around the rings, hydrogen atoms and hydroxyl groups are bonded [3]. The two rings are bound together by an oxygen bridge [3]. The central bridge may be broken when the β-Gal binds to either end and a water molecule reacts with the oxygen in the middle [3]. This process is known as hydrolysis due to water’s interaction in the middle. What remains is glucose and galactose [10].
The purpose, however, of β-Gal in this experiment is not for the E. coli to hydrolyze lactose, but rather to hydrolyze the X-Gal compound that will be added along with the plasmid DNA. X-Gal is an organic compound with a structure similar to that of lactose [6]. The break down of X-Gal by β-Gal is also similar to that of lactose: producing two simpler molecules, galactose (the same as in lactose) and 5-bromo-4-chloro-3-hydroxyindole [6]. The latter is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product[6]. Thus the hydrolyzation of the X-Gal compound into its constituent parts produces a blue coloring evident in the cells if transformation is successful [6].
3. Escerichia coli:
Theodore Escherich first described E. coli in 1885 when he isolated it from feces of newborns and for much time, until it was found to be the cause of diarrhea infection in infants in 1935, it was thought to be only a commensal organism of the large intestine [16]. E. coli and its relatives are known as “enteric bacteria” because they live in the digestive tract, particularly the intestines, of humans and other animals [16]. In close relation to E. coli and among the enteric bacteria is Shigella, the bacterial cause of dysentery, and Salmonella [16]. E. coli is a member of the family Enterobacteriaceae: this family is made up of gram-negative, nonsporeforming, rod-shaped bacteria that are often mobilized by flagella [16]. They grow well either in presence or lack of oxygen and thus metabolism may either be by respiration or fermentation [16].
E. coli is a very versatile bacterium, in the laboratory it may grow with glucose as its only organic constituent and may still produce all of the molecular components to make up the cell [16]. Wild-type E. coli is even more versatile as it has no growth factor requirements and can even grow under anaerobic or aerobic conditions [16]. In its natural environment, as well as in the laboratory, E. coli can respond to changes in temperature, pH, osmolarity and others [16]. In response to changes to temperature and osmolarity the cell might regulate the diameter of pores [16]. It can open up pores to accommodate larger substances or tighten pores to exclude inhibitory substances, like bile salts [16]. In the presence or absence of gases around them, the cells may swim toward or away from them [16]. It may stop swimming altogether and grow fimbriae to attach itself to a surface to keep it from moving. E. coli may also survey the environmental contents and “decide” (determined actually by genes within the cell) which enzymes it needs to breakdown and use these available solutions [16]. E. coli will thus not produce enzymes it does not need and will always produce the ones it does [16].
4. Ultraviolet Radiation:
Ultraviolet radiation is defined as that portion of the electromagnetic spectrum between X-rays and visible light, or from 40-400nm [20]. Ultraviolet Radiation is categorized by three major categories: Near Ultraviolet, NUV, Far Ultraviolet, FUV, and Extreme Ultraviolet, EUV, or Vacuum Ultraviolet [12]. Near Ultraviolet light, UV light that is closest to visible light, is then further divided into three sections that are more well known: UV-A radiation (320-400 nm), UV-B radiation (290-320 nm), and UV-C radiation (220-290 nm) [8][20]. The sun is our primary natural source of UV radiation. Artificial sources include tanning booths, black lights, curing lamps, germicidal lamps, mercury vapor lamps, halogen lights, high-intensity discharge lamps, fluorescent and incandescent sources, and some types of lasers [20]. Stratospheric oxygen or ozone absorbs 97-99% of the sun’s high frequency UV light with wavelengths between 150-300 nm [15]. It is the remaining percentage that sneaks through our atmosphere that are famous for causing sunburns and freckles [20].
UV-A radiation has the longest wavelength and lowest frequency and is therefore the least harmful. However, because of its low energy it passes relatively easily through the protective ozone guarded atmosphere and is the most encountered UV radiation on the planet [20]. This is the “tanning” radiation, in which it produces an initial darkening of the skin, followed by erythema if the exposure is excessive [20]. UV-A is needed by humans for the synthesis of vitamin D however over exposure has been linked to toughening of the skin, suppression of the immune system, and cataract formation [18]. UV-A is often called black light and most phototherapy and tanning booths use UV-A light [20].
UV-B radiation has a shorter wavelength and higher frequency than UV-A, but is longer in wavelength and lower in frequency than UV-C. Strangely enough though, UV-B typically does the most damage on Earth [20]. This is because it has enough energy to cause photochemical damage to cellular DNA, but does not have enough energy to be completely absorbed by the Earth’s ozone layer [20]. Like UV-A, UV-B is needed by humans for the synthesis of vitamin D and, like UV-A, negative side effects occur including: erythema (sunburn), cataracts, and development of skin cancer [20]. Most of UV-B radiation is blocked by the Earth’s ozone layer, but increased concern has come about over whether the reductions of the ozone layer could increase the prevalence of skin cancer [20].
UV-C is almost never observed in nature because it is completely absorbed into the ozone layer due to its high energy, as are Far and Vacuum Radiations [20]. UV-C radiation has the shortest wavelength and the highest frequency making for the most potent combination. Germicidal lamps are used in areas like dentistry and medicine for their ability to kill bacteria and therefore sterilize materials [20]. This is the “burning” radiation and “sunburn” is the least of your worries from accidental exposure. Accidental exposure may cause: corneal burns, commonly termed welders' flash, and snow blindness, a severe sunburn to the face, though these generally clear up in a day or two, they may be extremely painful [20]. UV-C in humans is absorbed by the outer and dead layers of the epidermis if exposure is not extreme or threatening [20].
5. DNA Repair:
DNA (deoxyribonucleic acid) is a linear polymer comprised of four different nucleotides, thought of as the building blocks of life, and each nucleotide is composed of three different parts: (1) a nucleotide base pair, either a purine (Adenine, A, or Guanine, G) or a pyrimidine (Cytosine, C, or Thymine, T); (2) a sugar, called deoxyribose on the count of it missing one hydroxyl group compared the ribose molecule of RNA; (3) a phosphate group binding to the sugar of the nucleotide above it (or below in the antiparallel direction) [1]. The nitrogenous base determines the identity of the nucleotide and thus their order is the makeup of the entire genetic sequence, a series of four letters that governs life. Inside the cell, DNA contains all of the information necessary for the cell to function, but the job of translating it and making it work in the cell is for RNA (ribonucleic acid) [1].
DNA within the living cell is subject to many chemical alterations and if the integrity of the genetic material is to remain sound, than these alterations must be corrected, a failure to correct an alteration becomes a mutation that may be heritable [9]. There are many agents that cause damage to the genetic structure including: certain wavelengths of radiation, highly reactive oxygen radicals, chemicals in the environment, chemotherapy [9]. Ionizing radiation, such as gamma and x-rays, and ultraviolet radiation, especially UV-C and –B, are the most common causes of damage to DNA by radiation [9]. Many hydrocarbons, including some found in cigarette smoke and some plant and microbial products, are some environmental chemicals found to cause DNA damage [9].
All of these damagers work in specific ways and produce certain kinds of DNA damage. The four amino groups (A, T, C, or G) could be modified at any point along the DNA chain, for example a base pair could be lost entirely (“deamination”) converting a C to a U (normally only found in RNA) [9]. The most common type of damage is done by mismatching of bases due to failure of proofreading during DNA replication. Damage may also occur from complete breaks in the backbone and those may be either single or double strand breaks. Finally, damage may occur along the DNA double helix due to crosslinks, covalent linkages formed between bases either between base pairs on the same strand or between pairs on opposite strands [9].
Due to the multiple types of Damage along the DNA strand there are multiple pathways of DNA repair. Direct chemical reversal of damage occurs in response to the spontaneous addition of a methyl group to a C base pair followed by the deamination of a T base pair [9]. Most of this kind of damage is fixed easily by glycosylases that do not require the excision of any base pairs [9]. Base Excision Repair (BER) occurs in response to single base pairs that are either missing or mismatched. In this repair the sugar-phosphate backbone is removed along with the base and DNA polymerase β fills the gap [9]. Nucleotide Excision Repair (NER) responds to the same kind of repair and in a similar way to BER but instead takes out large patches of genetic material even though there may be only one “bad” base pair [9]. Mismatch Repair (MMR) deals with the correcting of mismatches to normal base pairs, in other words, MMR deals with the failure to maintain normal Watson and Crick pairing of A to T and C to G [9]. Single strand breaks are repaired by the same enzyme systems used in Base Excision Repair [9]. Double strand breaks, however, are much more complicated and have two mechanisms for repair: direct joining and homologous recombination [9]. Direct joining involves using enzymes to recognize the break and bring the two pieces together [9]. Homologous recombination utilizes information stored on the sister chromatid or the homologous chromosome to replicate the DNA over in the correct fashion [9].
DNA repair is a necessary and vital process for all life as it maintains the integrity of the genetic material and thus the integrity of life. It is a major defense against environmental damage to cells and is therefore present in all organisms examined including bacteria, yeasts, drosophila, fish, rodents, and humans [19]. DNA repair is involved in the process to minimize cell killing, mutations, replication errors, persistence of DNA damage, and genomic instability [19]. Abnormalities in these repair processes lead to certain cancers directly and the development of others, and aging [19].
