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Applications of Highly Competent Cells

Discuss about the Analytical Applications Of Microbial Fuel Cells.

Highly competent cells are those that have transformed thereby bearing essential biochemical characteristics (Liu et al., 2014). Viable examples of the competent cells include E. coli DH5α and BL21 (DE3) cells. A biotechnology company should manufacture and sell the cells due to their multiple applications in the scientific field. The ability of E.coli to undergo biological transformation enables scientists to use the bacterial strain as a host for DNA manipulation. E. coli strains can also act as a carrier for protein expression. The transformation process applies the plasmids to facilitate protein expression and DNA manipulation to produce desirable products. Companies should produce highly competent cells since they can readily take an exterior gene and express its genotypic characteristics.

The scientists can apply the highly competent cells in other areas apart from gene expression and manipulation. The researchers can use the cells in DNA amplification and cellular analysis. Moreover, competent cells aid in genome editing and epigenetic (Hsu, Lander, & Zhang, 2014). Recent research has also shown that highly competent cells are necessary for glycobiology and DNA sequencing. Furthermore, the cells aid in DNA modification and synthetic biology.  Researchers have also indicated that they need the competent cells in the cloning process. Therefore, the vast range of application forces the biotechnology companies to manufacture and sell the competent cells.

The highly competent cells exist in numerous varieties of both shapes and size. Therefore, customers can choose the type they want according to the task that they are preparing to accomplish. Scientific activities like cloning and synthetic biology require the application of E.coli DH5α (Tschirhart et al., 2017). On the other hand, tasks such as DNA modification and sequencing require the use of BL 21 (DE3) cells. The preparation of E. coli can occur regarding single or multiple vials. Therefore, the significant variety of the competent cell types complements numerous biotechnological research methods.

During the shipment of highly competent E. coli cells, there is lack of surcharged ice on the cellular surface. Therefore, storage of the cells is more comfortable, and the cells are durable (Smargon et al., 2017). The manufacturers include a suitable media for outgrowth. The inclusion of the media allows room for the bacteria to grow and multiply into numerous colonies. Therefore, the buyers should not purchase large quantities of cells due to the possibility of overgrowth. Moreover, the purchasers can use the remaining colonies to perform other processes. Buyers should apply the highly competent cells since they are economical to the expenditure.

Production of High Competent Efficiency Cells

The competent cells lack animal products. Therefore, the competent cells are pure and natural to isolate (Tajima, Ichiyanagi, Yoshihara, & Hirokawa, 2016). Highly competent cells simplify the process of transformation in different animals. The cells apply to a wide range of animals hence they are universal. The competent cells readily bind with varying plasmids with minimum rejection rates. They exist in small sizes thus require minimal space for the process of transformation. E. coli strains record zero chances of errors during cell manipulation process. Furthermore, cloning and DNA modification occur best through the use of highly competent cells. Biotechnology firms should manufacture and cells highly competent cells to maximize their profits.

Highly competent cells refer to colonies that have an elevated competency (Bird et al., 2015). Furthermore, the researchers can quickly transform the cells. The ease of transformation of the cells determines their quality. The cell types that require a lot of time to transform are lowly competent cells. However, those that need a short time to undergo the biological process are the highly competent cells. Examples of high competent cells include the E. coli strains of BL 21(DE3) and DH5α colonies.  Therefore, the production of highly competent cells target colonies that require the shortest time to undergo the process of transformation.

Competent cells can quickly pick up a foreign genetic material and make a DNA product of exogenous characteristics. Strains of Escheria Coli are highly competent due to their high affinity for foreign DNA in the form of plasmids (Nielsen, & Keasling, 2016). E. coli cells can undergo a repeated cycle of freezing and thawing without any alterations in the structure of the cell or the plasmid. Moreover, the adjustment in temperature does not affect the ability of E. coli and the plasmid to undergo the process of transformation. Therefore, the cells that readily combine with foreign DNA to form a hybrid are described as highly competent.

The first process of production involves the hybridization of the foreign DNA with the plasmid gene molecule. The process is cheap for a biotechnology company. The combination of the two sets of genetic materials yields a hybrid complex which is ready for the next process. The second phase involves driving the complex hybrid into the E. coli cells (Wang et al., 2016). The hybrid plasmid complex undergoes the processes of replication inside the cell-transforming. Moreover, the amplification process also occurs inside the viable cell. The methods of making a highly competent DNA are numerous and take little time to conclude. Scientists prefer the procedures that are neither tedious nor time-consuming.

Modifications in Hanahan's Method

A suitable process in the production is the Bacteriophage technique. The phage can survive under diverse temperature conditions. In the process, E. coli combines with bacteriophage DNA at temperatures below one degree Celsius (Green, & Sambrook, 2018).  The process requires the presence of fifty milligrams of calcium ions. The method is not limited to the uptake of plasmid DNA as it can also apply to chromosomal DNA molecules. The researchers should adjust the temperatures to 38º C from the 1º C. The improvement of the effectiveness of the process involves the inclusion of additional cations like manganese and magnesium. The investigators should also increase the incubation time inside the calcium chloride solution.

The scientists have to first prepare the strains of E. coli before the start of the experiment. The growth of E. coli occurs best at thirty-seven degree Celsius. The strains are easy to grow than other grams negative bacteria. The researchers must include manganese, calcium, and potassium ions in the growth medium containing the strains of the cells (Cuiv et al., 2018). The investigators should lower the temperatures to zero degrees Celsius before the addition of plasmid DNA into the colony containing E. coli. The combination of the two components also requires the presence of dithiothreitol and dimethylsulfoxide molecules. Furthermore, the presence of cobalt chloride with hexamine chains speeds up the speed of reaction.


The most suitable method of preparing highly competent cells is through the Hanahan's method. The first procedure involves the re-suspension of the bacteria into the growth culture (An, 2018). The re-suspension should be in a gentle manner in a third volume of the starting FSB solution. The next step is incubation for ten minutes on ice. After incubation, the researchers remove the samples and pellet it four degrees Celsius for five minutes. A suitable centrifuge or Eppendorf applies to pellet the E. coli.  The researchers then add DMSO thrice after every five minutes. The next procedure involves swirling the suspension gently. Storage then follows in aliquots of two hundred microlitres at negative eighty degrees Celsius. Hanahan’s method is universally accepted due to its efficiency.

The Hanahan's method can perform better when the biotechnologists consider certain modifications. The first alteration involves the temperature involved in the growth of E. coli bacteria. The original way suggests a growth temperature of thirty-seven degrees Celsius (Taylor, Denson, & Esposito, 2017). However, growing colonies at lower temperatures speed up their development. Sodium chloride should not be ever present in the growth medium. However, the nutrient should be absent or present depending on the strain of E. coli used in the process of transformation. The biotechnologists should include sodium succinate in their experiments. The modifications make the process economical.

The researchers should extract plasmid DNA from the E. coli after the growth of the bacteriophage. The investigators should use miniprep kits to obtain plasmids of desirable quality from strains of E. coli (Savilahti, & Rasila, 2016). The extraction process should take a minimum of thirty minutes and a maximum of forty-five minutes. The extracted plasmid DNA components should then undergo purification before its application. Preparation for plasmid isolation involves the addition of RNase to the original buffer. The researcher should then mix the elements thoroughly by shaking the container gently. The mixing forms the initial pre-isolation phase. The RNases get rid of RNAs to remain with DNA.

The second step involves warming the buffer that has undergone lysis. The procedure should occur at thirty-seven degrees Celsius to dissolve any remaining particles in the solution. The investigators should then add ethanol to the buffer and mix the solution gently (Juhas, & Ajioka, 2016). The next step involves the storage of used buffer at suitable temperatures. The researchers should allow the resolution of E.coli to stand overnight to allow for further growth. The plasmid DNA is now ready to undergo the process of purification before application. A centrifuge or an Eppendorf are useful purification tools. The process ensures a germs-free plasmid DNA for the process of transformation and other uses. The lysis opens up the plasmid for the attachment of foreign DNA.

The suitable microcentrifuge should ensure centrifugation at less than twelve thousand gravitational rotations per given time. The process of purification should occur at temperatures that favor the growth of the E. coli strains (Reddy et al., 2016). The researchers should heat the aliquots containing the buffer before centrifugation. The process of re-suspension of the pellet should follow centrifugation to ensure a homogenous solution. The researcher then adds lysis buffer and gently mix the solution. The next step is incubation for six minutes. Purification is necessary to isolate a plasmid DNA for efficient molecular processes. The process of incubation allows for the growth of the transformed E. coli.

 The cells include notable E. coli strains such as BL21 (DE3) cells and DH5α. The cells produce a significant variety of end-user products due to their competency levels. Some of the notable products include RNA reagents and DNA markers (Andrade, Kroutil, & Jamison, 2014). The laboratory technicians use the reagents to isolate RNA from test samples. DNA markers serve as an indication of the presence of genetic materials in plants and animals. Other end products of competent cells include the ladders used in the Polymerase Chain Reactions (PCR). The ladders indicate both quantity and quality of harnessed genetic materials. Restriction endonucleases also emanate from the competent colonies. The enzymes cut the genomic material from the interior components to allow for transcription and translation processes. Therefore, competence cells produce useful biological products.

The products of the cells have a wide range of uses in the field of molecular biology. The first application involves cellular analysis (Wallden, Fange, Lundius, Baltekin, & Elf, 2016). The products are useful in determining whether a cell is highly competent or otherwise. The second usage is genetic cloning and genomic sequencing. Highly competent cells produce viable clones compared to the incompetent colonies. The production of competent cells has revolutionized the field of DNA sequencing. The cells are also useful in protein expression and purification. Competent cells enable researchers to identify hidden amino acid chains through the DNA markers. Furthermore, the isolation techniques allow biotechnologists to purify proteins (Abrevaya, Sacco, Bonetto,  Hilding-Ohlsson, & Cortón, 2015). The cells are essential in DNA amplification and RNA analysis. The PCR process depends on competent cells for the manufacture of the ladders and markers. The products of competent cells have a wide range of application in biotechnology.

Quality control (Q.C) refers to a protocol that ascertains that a particular product conforms to a pre-determined quality or needs of the consumers (Arrieta, Blackwood, & Glembotski, 2017). Quality assurance (Q.A) has a similar definition with quality control, but the two have a slight difference. Quality assurance is a protocol that gauges the suitability of a product at the previous levels (Gurley et al., 2016). On the other hand, quality control assesses the quality of a product after the manufacturing process. However, in most industrial setups, the workers use the two quality words interchangeably. Therefore, there is a clear-cut difference between Quality assurance and quality control.

The procedure for producing high competent efficiency competent cells requires both QC and QA to ensure the production of viable cells. The laboratory results for E. coli DH5α indicates a neat DNA in lanes one to four. Lane 5 has a dilution DNA, meaning that there was an omission of critical quality measures to ensure the production of neat DNA. Lane 6 has a neat DNA which fulfills the requirements of the quality parameters. Quality control parameters are essential at each step of producing competent cells (Arrieta, Blackwood, & Glembotski, 2017). Furthermore, the purification of plasmid DNA also requires checking to ensure the production of high-quality genetic materials.

The first step of plasmid isolation requires quality control checks to ensure that the DNA produced is pure. The action involves shaking the DNA pellets and leaving the solution to stand overnight under thirty-seven degrees Celsius. There is a danger that the DNA produced might not be enough for application in biological applications such as gene cloning (Gurley et al., 2016). The prevention of low yields of DNA involves the use of plasmids with the highest number of copies. Moreover, the researchers should provide viable conditions to enhance the growth of the colonies.

The investigators should also multiply the number of plasmids to prevent the production of low quantities of pure genetic materials. Moreover, the researchers can improve the amount of inoculated culture to facilitate significant yields (Ren et al., 2017). The separation method should belong to enhance the level of plasmid yield per culture. The researchers should eliminate media then re-suspend the pellets. Furthermore, ensuring that the process of suspension is complete leads to the production of high-quality plasmids. The researcher should use few cells in case of a viscous lysate. Therefore, the first step of plasmid isolation is the initial quality check in the production of competent cells.

The second procedure in the production of competent cells is an addition of buffer solution to the DNA pellets. The researcher should gently mix the solution by shaking it. The quality control issue in the stage is a possibility of denaturation of plasmid DNA. Prevention of denaturation involves the regulation of incubation time (Ren et al., 2017). The investigators should ensure that the duration of incubating the sample does not exceed five minutes. Furthermore, the temperatures must be suitable for the development of the E. coli strains in the experiment. The biotechnologists should regulate the incubation temperature and time to ensure high-quality plasmid DNA.


The third procedure involves the addition of the second solution followed by gentle swirling for five minutes. There is a possibility of DNA contamination at this step. Quality control measures are essential to prevent contamination (Cedeño, Pauwels, & Tompa, 2017). The production of impure genes interferences with molecular processes such as genomic sequencing and cloning processes. The initial measure to eliminate contamination is through gentle inversion of the reaction tubes to prevent the entry of undesirable micro-organisms. Furthermore, the incubation period should be under five minutes. The RNA can also attract impurities as a result of prolonged incubation and rough shaking of the reaction container.

The fourth procedure involves the addition of nine hundred microlitres to the buffer solution containing the plasmid under purification. Improper handling of the step leads to inconsistent enzyme activity (Hajirezaei, Darbouy, Rasouli, & Kazemi, 2015). The enzymes are the significant factors running the purification reaction. The hindrance in the activities of the catalyzers hampers the production of desirable cells for gene transformation. The ideal solution of ensuring non-stop enzymatic function is through effective centrifugation. The procedure dries the reaction column thereby removing the remaining buffer. The investigators should provide proper centrifugation to speed up the activity of the enzymes.

The fifth step in plasmid isolation is gently removing the supernatant. Quality issues can arise as a result of the reduced speed of the reaction column (Hajirezaei et al., 2015). The primary cause of the matter is the loose attachment of the manifold to the source of the vacuum. Another possible reason for slow reaction speed is opening of luer parts that the researcher did not use in the experiment. The first step of ensuring quality plasmid is through tightening the manifold to the source of vacuum. Moreover, the investigators should close the extensions that they did not use during the experiment. The reaction column should maintain a reasonable speed to ensure efficient production.

The quality control issues require a periodic timing to ensure efficiency. Moreover, the scientists should carry out a regular check-up to ensure that the end products are in tandem with the choices of the customers (Çimen et al., 2015). Both the production and the purification steps require a keen inspection to ensure quality. The first step in the Hanahan's method is gentle re-suspension of the bacteria. The researchers should carry out the process in a third volume of the starting solution. Elevation in the quantity of the amount interferes with the quality of plasmid DNA produced. Furthermore, a rough shaking of the reaction container denatures the genetic molecules.

The second step in the Hanahan’s procedures involves incubation for fifteen minutes on an environment containing ice. The researchers must observe the incubation time not to exceed fifteen minutes (Çimen, Y?lmaz, Perçin, Türkmen, & Denizli, 2015). Furthermore, the biotechnologists must ensure that the environment is ice-cold to provide an optimal environment for enzyme activity.  The pelleting process should take eight minutes and at four degrees Celsius. An extended period interferes with the quality of the genetic material. Furthermore, the application of less or higher than four degrees Celsius can denature the DNA and hamper enzyme action. Therefore, the researchers should observe strict temperature and time requirements.

References

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