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Selection of Species for Synthetic Drug Creation

Discuss about the Microbiology for Synthetic Biology Applications.

The science of synthetic biology varies from the conventional genetic engineering. As stated by Paddon et al. (2013), Synthetic biology can be defined as the construction and designing of biological entities for useful purposes. This field of science can be used to produce synthetic microbes which are specifically designed to perform a particular task like production of drugs and controlling pollution. In the arena of medicine, problems still exist in the clinical treatment of several chronic diseases. One of the most frequently occurring disease that need to be addressed is malaria. In this essay the designing of a synthetic microbe to produce a drug for malaria will be illustrated. In order to develop the synthetic microbe the novel gene that was separated is artemisinin and it was selected from Artemisia annua species (Ashley et al. 2014).

As discussed by Kent et al. (2012), the species selected for creation of the synthetic drug was Mycoplasma mycoides. These organisms were extracted from ruminants. This strain of Mycoplasma was used as the template. These organisms lack cell wall and for culturing and growth it requires steroid. These organisms grow better in broth and mycoplasma agar and can survive for longer duration at 45 degrees centigrade. According to Lehmeyer et al. (2016), the Mycoplasma mycoides require sterols for the stability of their cytoplasmic membrane. Since its difficult to culture Mycoplasma strains in other microorganisms because it needs sterols to function, therefore the host organism for designing the synthetic microbe could be Mycoplasma yeatsii. These species have a common host and body site with other Mycoplasma species found in ruminants. Since the plasmid selected would be hosted by another species of the same organism even without cell wall they can survive. Moreover, these microorganisms although lack cell wall they are unaffected by several antibiotics such as beta-lactam or penicillin (Karas et al. 2014).

As stated by Moe-Behrens et al. (2014), Plasmids are significantly useful cloning vectors that can be used to isolate, purify and multiply inside the bacterial cells. The plasmid genome of the M. mycoides pADB201 (NC_001382) should be used after isolating it. pADB201 encodes two proteins orfA and orfB. The origin of plasmid is composite. In Such rolling circle plasmids, the single stranded origin is the initiation site of the lagging strand. This is important for conversion of the single stranded molecule into double stranded molecule. The plasmid vector would be designed in such a way that the vector would contain two replication origins, multiple cloning site which will consist of a restriction cut site and a antibiotic resistance site (ampicillin resistant site). The various restriction sites that would e incorporated are BamHI, HaeIII, Hind III, EcoRI and AluI. Addition to this a Lac Z site will be inserted into the multiple cloning site in order to identify the transformants. The virulent gene would be removed in order to prevent disease causing affect in the animal to be tested on (Karas et al. 2014).

Plasmid Vector Design

As discussed by Gourgues et al. (2016), “Transformation” is the process of introduction of exogenous genetic material into the bacterial cell. In this experiment the plasmid DNA would be introduced into the Mycoplasma yeatsii. The plasmid mediated transformation of the Mycoplasma mycoides would follow certain steps. Firstly, the plasmid vector would be cleaved using EcoRI endonuclease at the restriction site to produce sticky ends. According to Ariey et al. (2014) this the foreign DNA that is artemisinin which would be extracted from Artemisia annua species will also be cleaved using the same enzyme at the AATT sites. The insertion of the gene of interest will be done. The ligase enzyme initiates the formation of the phosphodiester bond. The ligase enzyme will catalyzes the annealing of the plasmid genome with the specific gene of interest. The whole process will be conducted in media containimg NEB buffer (Fischer et al. 2012). The metabolism of Mycoplasma yeatsii is similar to other Mycoplasma where the main organic compounds are glucose and mannose. Therefore, sterol and arginine are the two key growth factors for the survival off this organism. Moreover, this organism is aerobic in nature, so requires the presence of oxygen to multiply. Lastly, they do not decompose urea.

According to Dordet-Frisoni et al. (2013), the host cell is made competent so that the plasmid can enter the cell. There e three steps that should be followed in order to continue transformation. Firstly, Calcium Chloride should be added to the medium in order to enhance the introduction of the plasmid DNA into the host cell. Secondly, the cells are “heat shocked” and thirdly, the cells are incubated in the nutrient broth for a short duration. Lastly, the broth containing the transformed cells is plated in the agar medium. The plasmid vector containing the gene of interest would replicate utilizing the host machinery and would result in the transformants. In order to check whether the transformation has completed successfully or not screening through the process of blue white colonies are done. As discussed by Fischer et al. (2012), the medium would be cultured with X-Gal and IPTG. If the gene of interest gets inserted within the Lac Z site then the X-Gal will remain inactive producing a white colony. On contrary, if the gene of interest inserts outside the Lac Z gene or does not get inserted at all then the Lac Z gene will be activated by IPTG and the X-Gal will be broken down forming blue colonies (Kent et al. 2012).

Plasmid Mediated Transformation of Mycoplasma yeatsii

The synthetic microbe that is formed can be used for preparing drugs against malaria. The synthetic genome would contain nucleic acid biosynthesis, ribosomal operon, transporters, DNA repair tools. The operon system of the synthetic microbe would contain NBOPr which is a substrate inducible promoter, NBO1-NBO3 is the genes that encode the biosynthetic pathway and NBO4 is the product transporter (Kent et al. 2012). The noble gene artemisinin mimics the CR-trigger hydrogen peroxide and nitric oxide to induce antioxidative networks and mitigate the oxidative stress. Thereby, direct the metabolic conversion of anabolism to catabolism, extending the life expectancy and metabolic functionality in the host.

There are certain genes that are developed or constructed by various evolutionary processes. Out of these genes, several genes produce duplicate copies of the total gene while the others rearrange the existing genetic molecules and are termed as novel genes. According to Karas et al. (2014), the main function of these novel genes are that they contribute to unique phenotypes leading to character differentiation in organisms. In this experiment artemisinin gene will be used as a novel gene to contribute in the cure of malaria. In this experiment the novel gene will be regulated using plasmid mediated transformation which will be used to create a drug in order to test the clinical applications of the drug against malaria in mice (Fischer et al. 2012).

Most microbes consist of a gene that secretes a protein called CRISPR. This particular protein has the ability to bind to a specific recognition site in the plasmid and cleave it. This is a kind of defense mechanisms in microbes that cleaves a foreign gene as soon as it enters the host cell. Thus, during the designing of the vector the recognition site for this protein should be removed via ligase enzyme in order to maintain the gene of interest. This process would help the plasmid to be stable and grow in the host cell.

Regualatory mechanism

Diagram 1: Regualatory mechanism

Components of the Synthetic microbe

Diagram 2: Components of the Synthetic microbe

In the developing and technologically superior method of synthetic biology there exist potential high risks which if used in the form of a bio-weapon. First of all, the synthetic microbe designed can result in negative result in the host cell or organism in which the synthetic drug would be applied. Secondly, there are ethical risks associated with the use of the microbe by the society as a treatment mode.

Construction of Synthetic Microbe for Malaria Drug Production

As stated by Dordet-Frisoni et al. (2013), Synthetic biology represents an effective area of science that combines the biological science with the systems engineering. According to the evidences provided the synthetic organisms have shown successful results in the clinical field involving studies, diagnosis and treatment. The ethical arguments in view of patenting extend to the reality that monopolization of biological substance can cause to reduce in contact of such material to the community. Since e pathogenic nature of the microbe will be eliminated during the experiment the use of the organism can be a safe choice. The synthetic microbe cannot be first tested in human beings rather it will be used in mice due to ethical issues of Mycoplasma that causes tuberculosis and other serious diseases. Moreover, the use of the plant gene artemisinin can be used as an environment control measure. This synthetic microbe can be patented due to the use of an innovative novel gene into the construction of a synthetic microbe. The rising field of synthetic biology is seasoned for law appraisal and improvement, both in US and Australia. There has been a propagation of patents in this arena, with the possible for important impact on health, the environment and the economy. For example, Nygone, which is a microplastic filter designed to degrade nylon (Karas et al. 2014).

References:

Ariey, F., Witkowski, B., Amaratunga, C., Beghain, J., Langlois, A.C., Khim, N., Kim, S., Duru, V., Bouchier, C., Ma, L. and Lim, P., 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature,505(7481), pp.50-55.

Ashley, E.A., Dhorda, M., Fairhurst, R.M., Amaratunga, C., Lim, P., Suon, S., Sreng, S., Anderson, J.M., Mao, S., Sam, B. and Sopha, C., 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. New England Journal of Medicine, 371(5), pp.411-423.

Dordet-Frisoni, E., Baranowski, E., Barré, A., Blanchard, A., Breton, M., Couture, C., Dupuy, V., Gaurivaud, P., Jacob, D., Lemaitre, C. and Manso-Silván, L., 2013. Draft genome sequences of Mycoplasma auris and Mycoplasma yeatsii, two species of the ear canal of Caprinae. Genome announcements, 1(3), pp.e00280-13.

Fischer, A., Shapiro, B., Muriuki, C., Heller, M., Schnee, C., Bongcam-Rudloff, E., Vilei, E.M., Frey, J. and Jores, J., 2012. The origin of the ‘Mycoplasma mycoides cluster’coincides with domestication of ruminants.PLoS One, 7(4), p.e36150.

Gourgues, G., Barré, A., Beaudoing, E., Weber, J., Magdelenat, G., Barbe, V., Schieck, E., Jores, J., Vashee, S., Blanchard, A. and Lartigue, C., 2016. Complete genome sequence of Mycoplasma mycoides subsp. mycoides T1/44, a vaccine strain against contagious bovine pleuropneumonia.Genome announcements, 4(2), pp.e00263-16.

Karas, B.J., Wise, K.S., Sun, L., Venter, J.C., Glass, J.I., Hutchison III, C.A., Smith, H.O. and Suzuki, Y., 2014. Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides.Frontiers in microbiology, 5.

Kent, B.N., Foecking, M.F. and Calcutt, M.J., 2012. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii. Journal of microbiological methods, 91(1), pp.121-127.

Lehmeyer, M., Kanofsky, K., Hanko, E.K., Ahrendt, S., Wehrs, M., Machens, F. and Hehl, R., 2016. Functional dissection of a strong and specific microbe‐associated molecular pattern‐responsive synthetic promoter. Plant biotechnology journal, 14(1), pp.61-71.

Moe-Behrens, G.H., Davis, R. and Haynes, K.A., 2014. Preparing synthetic biology for the world. Synthetic biology applications in industrial microbiology, p.77.

Paddon, C.J., Westfall, P.J., Pitera, D.J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M.D., Tai, A., Main, A., Eng, D. and Polichuk, D.R., 2013. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature,496(7446), pp.528-532.

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