Exploring Genetic Engineering

Introduction

Genetic Engineering is referred to the process of modification of an organism at generic level through the use of biotechnology. The modification at the genetic level which can be done through this process includes insertion of new DNA in the host genome, removal of genetic material using specific enzymes and gene targeting for development of a recombinant (Knott and Doudna, 2018). For those seeking specialised assistance, biology dissertation help can provide valuable insights into these complex processes. In this assignment, the detailed process involved in genetic engineering is to be explained. Moreover, two examples of genetic engineering which is production of Insulin and Hepatitis B vaccine are to be discussed by informing the process involved. Further, the advantages and disadvantages in relation to genetically engineered insulin and hepatitis B along with ethical considerations are to be explained.

Process of Genetic Engineering

The implication of foreign DNA as the recombinant DNA vectors which are modelled through molecular cloning is the most frequent method used in genetic engineering. The molecular cloning in genetic engineering is the process of formation of multiple exact copies of genetic information, expression of genes and studying specific genetic material (Shi and Vickers, 2016). In molecular cloning in genetic engineering, at first, the DNA fragment to be replicated is received from a donor. The DNA fragment is initially inserted into a plasmid so that it can be modelled in a form which allows it to be expressed or copied (Fan et al. 2019). A plasmid which is also known as vector in genetic engineering is a circular DNA of small size which independently replicates their chromosomal DNA within the bacteria.

In genetic engineering, during cloning, to insert a DNA fragment of desire the plasmid molecules are seen to act as a vehicle for the process (Berteroet al. 2017). The plasmids naturally occur within the bacteria such as E.coli and are frequently used in genetic engineering because of their ease of acceptance of foreign DNA fragment for replication. This is because they have small DNA sequences which can be easily cut through restriction enzymes for insertion of DNA for multiplication (Kaufman and Kirakosyan, 2016). The restriction enzymes are known as restriction endonuclease which has the role to identify specific DNA sequences and cleave them in predictable manner as they are produced naturally by the bacteria to act in defence against any foreign DNA. Most of the restriction endonuclease develops staggered cuts in DNA strands so that the cut ends are 2-‘to 4’nucleotide single-stranded sequence overhangs. The restriction enzyme detects a 4' to 8" nucleotide sequence which is palindrome meaning the sequence reads the same both back and forth. (Refer to Diagram) The staggered cuts made in the palindrome sequence are found to have complementary overhangs. Since the complementary overhangs show ability to stick together, they are known as sticky-ends (Mruket al. 2019; Kocioleket al. 2018).

The complementary sticky ends are created in plasmid and foreign DNA by using the same restriction enzymes for both of them. This is because it would help the foreign DNA when inserted in the plasmid to for annealing. The annealing is done through the help of DNA ligase which helps the sticky ends to permanently come together. (Refer to Diagram) After this, the bacteria which accept the plasmid recombinant are unable to produce enzymes from the gene fragment. These bacteria are also found to have a gene resistance for antibiotic ampicillin that is used as the original plasmid. Therefore, to identify the bacteria which have taken up recombinant plasmid are produced on ampicillin plate in genetic engineering where the ampicillin kills any bacteria those have not taken the plasmid, in turn, leaving the culture to have only bacteria which are required to be grown to get the recombinant genetic product (Wang et al. 2020).

The genetic engineering of products is also done through reverse transcription in which a DNA strand is created from an RNA template. The reverse transcriptase-polymerase chain reaction (RT-PCR) is used for amplifying RNA targets like HIV, influenza and others. In this process, initially, the RNA is extracted from the cellular body and is added to the mixture which contains reverse transcriptase enzyme along with primer for target and nucleotides (Pavlovicet al. 2017). The tRNAprimer detects the target on the RNA and hybridises complementary part on the RNA known as primer binding site (PBS). The reverse transcriptase present then initiates to add DNA nucleotides from the 3’ ends of the primerfor synthesising complementary DNA to the U5 and R region of viral RNA. The U5 is non-coding region and R region is direct repeat at RNA end which are degraded by the RNAse present in restriction enzyme. The RNAse H degrades the majority of RNA and leaves behind the PP sequence and helps the synthesis of the second strand occur on the PP sequence for double-stranded DNA formation. After a complete DNA strand is formed, it is incorporated in gene of the host through retroviral integrase which allows the insertion of DNA into the host chromosomal DNA (Baskar and Ramalingam, 2018). (Refer to Diagram)

The PCR (Polymerase Chain Reaction) in which a single DNA sequence is amplified in different magnitude for assisting in the process of genetic engineering of products includes four steps. The first step is DNA denaturation in which the hydrogen bonds forming the double-stranded DNA are broken through heat to form single strands of DNA. In the second stage, specific primers are chosen based on the target sequence to be replicated and they are annealed to the complementary sequence of single stranded DNA under certain temperature (40-65˚C). After annealing the primers, the temperature is increased and Taq DNA polymerase is used as enzyme for replication of the DNA strands. At the end of PCR, two identical newly formed DNA strands are present and they are repeated to form more strands (Pjevac et al. 2017).

Examples of Genetic Engineering

Insulin

In order to genetically engineer insulin, the reverse transcriptase process of molecular cloning is followed. Insulin is the hormone produced by the human pancreas for allowing the body to use glucose from carbohydrates in the food as energy to support effective functioning and restore glucose for future use by the body (Rajaeiet al. 2017). In the initial step, the extraction of human insulin is done from the pancreatic cell and isolation of the insulin-producing gene is done. The vector or plasmid used in the process is Escherichia coli and the restriction enzyme used for developing complementary sticky ends on the plasmid and DNA of the insulin gene is EcoR1 endonuclease (Babaeipouret al. 2018). The E.coli is used for genetic production of insulin as it allows economic and rapid production of recombinant compounds and proteins (Abdulkareem, 2019). The EcoR1 forms 4 nucleotide stick ends that are with 5’end overhangs of AATT indicating the recognition sequence of the nucleic acid where the enzyme is going to make the cleave is g/AATTC that would have CTTTAA/G as a palindromic complementary sequence. This informs that the EcoR1‘srestriction site is 5’-G/AATTC-3’ and 3’-C/TTTAAG-5’. A temperature of 37˚C is to be maintained during the action (Aziz et al. 2016).

After cleavage, the insulin DNA with sticky ends is inserted inside the E.coli where it forms recombinant DNA. The insertion is done by the side of lacZ gene as it is easier to be found and cut. The newly formed recombinant DNA is then transported in the fermentation task where they multiply through mitosis each expressing insulin gene. After multiplication, they are taken out from the tank and multiplied cells are broken open for extracting their DNA. The common way used is implanting lysosomal mixture to digest the cell wall followed by addition of detergent mixture to separate fatty cells exposing the bacterial DNA (Wong, 2018). The DNA is further treated by cyanogen bromide which is the reagent that breaks protein chains leading to separate insulin chains from the DNA. The insulin chains are then mixed to be joined together with the help of disulphide bonds that is done by organising reduction-reoxidation reaction. The batch is then placed in centrifuge and the DNA mixture is purified to have only insulin chains for commercial use (Landgraf and Sandow, 2016).

The advantages of genetically engineered insulin are that it is indistinguishable from the insulin produced within the body of humans making them be effectively used instead of medication that causes side-effects or allergic reaction (Yasmeen, 2018). This is because the genetically engineered insulin is detected by the body as its own product. The other advantage of genetically engineered insulin is that they can be produced in large quality within a small amount of time. Moreover, due to its immunogenicity, genetically engineered insulin is more effective for individuals (Baoet al. 2016). However, the disadvantage to be faced with genetically engineered insulin is that they are costly and some individuals using them reports of hypoglycemic complications (Caiet al. 2017). Thus, it cannot be easily availed by all diabetes patients in the society and may act to drastically lower blood sugar levels that increase the chances of further complications. There are ethical issues among different communities of using animal-produced insulin which are developed from pigs and other animals as the people in the community thinks it is not supported by their region as it so developed by harming the animals. In this case, the genetically engineered insulin is going to resolve the ethical issues (Yasmeen, 2018).

Hepatitis Bvaccine

The Hepatitis B vaccine is also developed through the process of restriction endonuclease and not through reverse transcriptase. The Hepatitis B virus (HBV) is a viral disease that causes severe chronic liver disorder. The DNA of HBV is found to be of 3Kb size with a large single-stranded gap. On infection of HBV in the body, it is found that HBV shows failure to multiply and cause infection to large cells or grow in culture cells. This mainly occurs due to its inhibition for molecular expression and creates hindrance in vaccine production (Yuen et al. 2018). Thus, the HBV’s recombinant vaccine was created through cloning of the HBsAg gene present in the virus by using yeast cells as the vector. The yeast cells are found to have complex membrane structure which allows it to easily secrete glycosylated protein. This has allowed it to be able to create autonomous replication of plasmid containing the HBsAg gene near the alcohol dehydrogenase (ADH) I promoter of yeast (Moshkaniet al. 2019).

There are 6 bp-long sequences present in HBsAg gene initiating from the AUG which causes synthesis of N-terminal methionine that is joined with the ADH promoter cloned in the PMA-56 yeast vector. The recombinant plasmid is later inserted into the yeast cells which causes transformed yeast cell arc that is multiplied within the tryptophan-free medium. The yeast cells which are transformed are selected for cloning to express HBsAg gene. The inserted genetic sequence express and creates particles which are similar to 22 mm particle of HBV as it is produced within the serum collected from blood of the HBV infected humans. The expressed HBsAg compounds are found to have similar structure along with immunogenicity related to isolated HBV-infected cells. The increased immunogenicity of them has made it able to be marketed in the form of vaccine for HBV infection (Tong and Revill, 2016; Yuen et al. 2018).

The advantage of genetically engineered Hepatitis B vaccine is that they are non-infectious and have the presence of defined virulence which causes them to show less side-effect on the body (Pumpens and Grens, 2016).The other advantage is that increased production of genetically engineered Hepatitis B vaccine can be made easily to provide support to a wide number of individuals (Pumpens and Grens, 2016). Thus, no scarcity of vaccine for HBV would be faced in the society. However, the disadvantage of genetically engineered Hepatitis B vaccine is that it is unknown regarding the way it may cat for different hosts. In addition, consequences to be faced with random insertion of vaccine constructs within cellular genome in target or non-target species within the environment is unknown (Lopez et al. 2017). The ethical issues regarding genetically modified vaccines like Hepatitis B are that it may cause unpredicted changes in the environment which could lead to the deteriorating health of the individuals (Lobaina and Michel, 2017).

Conclusion

The above discussion informs that genetic engineering is the process of creating modification in the genetic constructs of animals or humans. The process of genetic engineering includes the use of restriction endonuclease to develop complementary sticky ends in foreign and plasmid DNA for them to be recombined with the help of DNA ligase. In genetic engineering of insulin, the E.coli bacteria are used as plasmid or vector and EcoR1 is used as restriction enzyme. The advantage with its production is that its allergic reaction is avoided but the disadvantage is that they are high priced. In genetic engineering of Hepatitis B vaccine, the yeast cells are used as plasmid and HBsAg gene is used as recombinant gene to develop the vaccine. The advantage with its production is that it allows the vaccine to be widely available but the risk is that the way it may act in different hosts is not known.

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