Please supply inside informations of current and old research experience, e.g. holiday scholarship, undergraduate undertaking ( no more than 300 words ) .
I Have Successfully completed a preparation plan on “ DNA Fingerprinting by Restriction Fragment Length Polymorphism ( RFLP ) and Short Tandem Repeats ( STR ) ” organized by the “ Department of Biotechnology ” , Manipal University, Dubai Campus, UAE. Besides, while analyzing bioinformatics, I learnt how to entree the information archives of genomes and proteins, the tools that have been developed to work with these archives, and the sort of inquiries that these informations and tools can reply. I have emerged with a sense of optimism that the informations and methods of bioinformatics can make profound progresss in our apprehension of life, and the betterments in wellness scientific disciplines. While at undergraduate degree, I gained custodies on experience in techniques underpinning rDNA engineering such as readying of E.coli competent cells and synthesis of complementary DNA utilizing RT-PCR. I have besides dealt with the practical basicss of in vitro culturing of animate being and works cells and covered types of civilization, biological science of civilized cells, processs, methods and applications which made me familiar with unfertile techniques and media readying. I have had the chance to work with HeLa cell lines which involved their cryopreservation, subculturing, resurgence and quantitation utilizing a hemocytometer. All of this enabled me to larn about the rules of works and animate being cell civilization, commercial applications and assorted methods to bring forth the same. I have even completed a mini-project, working in braces, to sub-clone, mutate, express and sublimate the Green Fluorescent Protein, thereby deriving practical competency in techniques such as plasmid DNA readying and quantification ; limitation enzyme digestion ; site-directed mutagenesis ; PCR ; DNA sequencing ; Agarose Gel Electrophoresis ; SDS-PAGE and Western Blotting ; protein purification by Affinity Chromatography. Hence, I feel that I have gained a significant sum of practical research lab experience that would measure up me as a possible PhD applier to work with your celebrated research squad.
Outline the grounds why you wish to analyze for a PhD and the calling you intend to prosecute ( no more than 500 words ) .
I have ever wanted to accomplish something important. I am ambitious and I yearn to arouse myself to accomplish complex ends. I have ever loved to detect and larn about new findings. I have ne’er lost my naif involvement in scientific finds taking topographic point around the universe and so I believe I that would go a successful research worker. I possess an ageless enterprise within myself that propels me to research new things and larn more about them, and I love research, and therefore experience that a doctor’s degree will be perfect for me. I besides wish to better myself and my life. I want to heighten my abilities to grok and work out existent life jobs, increase my assurance and communicating accomplishments and develop competency that may take me to a better occupation. I think a PhD merely fits me. Some people are committed to prosecute a doctor’s degree and I feel I am one of them. I have ever worked on legion mini research undertakings as avocations, either in the signifier of look intoing workss or happening the grounds behind assorted cosmopolitan phenomena. I have a characteristic pursuit for cognition and an ardent preference for reading assorted books on specific subjects. In fact, I have had an ageless captivation about Biology.
The aim on completion of my PhD is to prosecute a really successful and a adept calling, that would let me to be recognized as an person who positively influenced the lives of all the people and every bit good as organisations that I was associated with. I intend to carry through all undertakings with a really positive attitude, a batch of devotedness, every bit good as an ardor to do a difference. I want to be exposed to a broad scope of research countries. In the initial period of my calling, I would wish to derive huge cognition and an ample sum of practical experience. My ultimate end would be to lend to up-to-date research in the Life Sciences and to develop fresh drugs and curative compounds for the benefit of world. I want to prosecute a fulfilling calling in the field of biological scientific disciplines, which is helping in the development of extended inventions in the medical, industrial and agricultural sectors that were earlier highly time-consuming and labor-intensive. Ideally, I would come in into a phase in my professional calling where I would be acknowledged as a well renowned research scientist. I would derive huge contentment and satisfaction by larning that my part to research has had a good impact on the being of all those affected. If I win in accomplishing all of my ends wholly, I should be able to get an copiousness of fear towards the latter portion of my life. I hope to take a long life to be able to slake my thirst for cognition and persevere in my pursuit for a fruitful calling in Biotechnology, therefore giving back to the society, and go forthing a permanent feeling on it for ages to come.
Word Count: 493
( a )
What is your research inquiry? ( no more than 100 words )
It is extremely desirable to show high degrees of recombinant human growing endocrine ( somatropin ) in bacteriums such as Escherichia coli, owing to its curative potency. However, the expressed protein frequently tends to aggregate within the bacteriums and form inclusion organic structures. This phenomenon impedes the isolation and purification of the functional recombinant protein, therefore restricting its handiness for medical applications. Attachment of an XTEN sequence as a merger spouse to the mark protein sequence could take to the production of soluble and biologically active somatropin, therefore cut downing the figure of arduous and time-consuming stairss employed in its purification and refolding processs.
Word Count: 100
( B )
Why do you believe this country of research is interesting? ( no more than 250 words )
Human Growth Hormone ( hGH ) is a 191 amino acid individual concatenation polypeptide of molecular weight 2,200Da, produced by the anterior pituitary secretory organ. It is used in the intervention of clinical conditions such as pituitary nanism in kids missing sufficient endogenous degrees of hGH to rectify short stature. Scientific grounds has besides facilitated blessing of its disposal for the intervention of pathological conditions such as chronic nephritic inadequacy, osteoporosis and terrible Burnss. Recently, it has besides been demonstrated to be effectual in the intervention of HIV related infection.
Such possible applications of the endocrine have generated widespread clinical and industrial involvement, and therefore, its demand as a curative protein has risen exponentially.
The coming of rDNA engineering has enabled industrial production of the endocrine by showing it in E.coli and this helped to partially get the better of its limited supply for clinical applications, as earlier the lone dependable beginning of the protein was the human pituitary secretory organ ( Gary and Headon, 1994 ) .
However, several jobs associated with the look of the protein in E.coli still exist. One such major job is its look in an indissoluble signifier in inclusion organic structures which makes its recovery as a functional protein significantly hard.
Application of a fresh experimental scheme, such as XTEN engineering, to heighten the solubility of the expressed recombinant protein and cut down the fraction of inclusion organic structures would afford cost-efficient and efficient recovery and purification of the curative protein, therefore doubtless simplifying and maximizing its large-scale production in a biologically active signifier, which would besides advance farther experimental surveies.
Word Count: 250
( degree Celsius )
How make you suggest to turn to this research inquiry? ( no more than 750 words )
Fusion of an XTEN sequence to a curative peptide exenatide has been shown to heighten the solubility and output of the affiliated protein spouse, when expressed in E.coli. A similar attack could be applied to somatropin, by using the experimental scheme outlined below:
1 ) . Preparation of Deoxyribonucleic acid fragments encoding somatropin and XTEN –
DNA sequence of somatropin would be prepared by utilizing the Phosphoramidite method of Chemical DNA Synthesis, based on its known amino acid sequence.
The cistron encoding the 864aa XTEN fragment would be synthesised as described in the literature. The XTEN and somatropin Deoxyribonucleic acid fragments generated would so be sequenced utilizing the dideoxy chain-termination method to guarantee their rightness.
2 ) . Fusion of somatropin and XTEN followed by ligation into a suited vector –
The DNA sequence for somatropin would be fused to a predating Deoxyribonucleic acid sequence encoding for a Tobacco Etch Virus ( TEV ) limitation site, utilizing adaptors/linkers or overlap PCR. Following this, the ensuing TEV/somatropin sequence would be fused to another predating Deoxyribonucleic acid sequence encoding for the C.thermocellum Cellulose Binding Domain ( CBD ) , once more utilizing overlap PCR.
The CBD-TEV/somatropin sequence therefore generated would be ligated into a pET30 derived XTEN look vector ( eg.pCW0359 ) by add-on of appropriate limitation sites, ( eg. Bsa1 and Bba1 ) to the vector and the merger sequence, resulting in the coevals of a CBD-TEV/somatropin-XTEN merger sequence. The resulting concepts would so be subjected to restriction enzyme digestion for analysis and confirmation.
3 ) . Transformation of appropriate bacterial cells followed by testing and choice of positive ringers –
The recombinant look vector would be transformed into E.coli DH5I± cells and subjected to DNA sequencing to guarantee fidelity, followed by transmutation into competent protein look host cells. Eg. E.coli Gold BL21 ( DE3 ) . Alternatively, E.coli cells could be made competent by electroporation or CaCl2 transmutation.
The cell civilization would so be plated onto LB-Kanamycin agar home bases, which would let growing of merely positively transformed cells as bacterial settlements. This would be possible as the Kantrex opposition cistron in the look vector would confabulate antibiotic opposition to the transformed cells.
Positive ringers would so be selected for confirmation by settlement PCR and agarose gel cataphoresis.
4 ) . Initiation of protein look in the positively transformed host cells –
The efficaciously transformed host cells would so be cultured in a Kantrex incorporating alimentary media placed in a shaking brooder to farther guarantee growing of merely transformed cells.
The growing of the civilization could be determined by mensurating its optical denseness ( O.D ) at 600nm. At this wavelength, 1 O.D would stand for 0.8A-109 cells/ml.
The cells would so be induced by IPTG to over show the merger protein. This would be achieved by adhering of IPTG to the T7 represser protein or its supplanting from the operator part of the bacterial DNA.
5 ) . Recovery of transformed cells and isolation of the merger protein –
Following initiation, the cells would be harvested by centrifugation and so lysed to let go of the merger protein of involvement along with other intracellular constituents.
The cells could either be ruptured automatically ( homogenization, micro-fluidisation, sonication ) or lysed chemically ( lysosyme + EDTA + SDS ) .
Removal of cell dust would be accomplished by differential centrifugation, which would give a clear lysate incorporating the soluble protein, as an expected effect of the alone features conferred by the XTEN sequence for maximal merchandise recovery.
The lysate would so be heated, followed by add-on of TEV peptidase to the sample, in order to split the CBD and TEV sequences from the merger protein.
6 ) . Purification, concentration and confirmation of the recombinant protein –
It would be prudent to use a lower limit of three chromatography stairss to obtain a virtually pure curative protein meant for parenteral disposal, as the presence of even trace sums of contaminations could take to adverse clinical reactions.
The protein sample would be applied onto ion-exchange and hydrophobic interaction chromatography columns to sublimate the mark protein on the footing of its net charge and hydrophobic features severally. The mark protein could so be concentrated by centrifugation and/or filtration.
The fidelity of the amino acerb sequence of the merger protein would be determined by N-terminal sequencing followed by non-reducing SDS-PAGE analysis to continue its native construction and gauge its molecular weight.
7 ) . Determination of physical belongingss possessed by the merger merchandise –
Mass Spectroscopy and Circular Dichroism would be employed to find precise mass of the protein and presence/absence of any secondary structural characteristics severally.
Gel Filtration Chromatography coupled with Multi-angle Light Scattering would so be used to find absolute molecular mass of the merger protein.
This scheme could potentially ease and hasten downstream processing of the curative recombinant protein.
Word Count: 750
( vitamin D )
What scientific considerations led you to take the research lab and supervisor for your research undertaking? ( no more than 150 words )