Cloning and expression of human | My Assignment Tutor

BS934 Assignment 2: Cloning and expression of human CHRAC1-EGFPIn answering this task, please, do not upload more than 3 A4 pages onto FASER by 20thApril 2021 16:00. Total maximal word count: 1000. Cite references or web sites whereappropriate.Please, structure and describe your strategy in sufficient detail so it could actually beimplemented. Explain your choices and rational briefly. Indicate sequences (e.g., primersequences) where applicable. Please, keep in mind that there is not ONE solution to thistask, but several options and you need only to identify one route. Please, decide on onlyONE route and describe it. What is important is that you briefly explain and justify yourchoices.We wish to test if human CHRAC1 is targeted to DNA repair sites in human cells (see TheACF1 complex is required for DNA double-strand break repair in human cells. Lan L, Ui A,Nakajima S, Hatakeyama K, Hoshi M, Watanabe R, Janicki SM, Ogiwara H, Kohno T, Kanno S,Yasui A. Mol Cell. 2010 Dec 22;40(6):976-87. doi: 10.1016/j.molcel.2010.12.003. PMID:21172662).To achieve this, you cloned the human CHRAC1 ORF cDNA into pCDNA3.1 so it would beexpressed as a fusion protein with EGFP at its C-terminus.Your tasks are as follow:1. Please, design primers for Sanger sequencing to verify the correct sequence(sequencing in both directions) of the CHRAC1-EGFP fusion cDNA in your expressionvector. Remember, that a stretch of quality Sanger sequence is only ~ 300 bp andthat good sequence only appears ~ 50 bp downstream of the primer. List the primersso they can be ordered from a company. You do not need to verify the sequence ofthe expression vector backbone itself, only CHRAC1-EGFP and the sequence where itis integrated in the vector.2. Please, show that your construct is expressed as a fusion protein without disruptionof the EGFP reading frame (e.g., show the nucleotide sequence of the transition fromCHRAC1 to EGFP and how this would be translated, e.g. 30 nucleotides up- anddownstream of the transition point and the translation into protein).3. Devise a strategy to transfect your expression construct into human cells. Outlinethe protocol that you would use and justify the choice of the method and protocol.To find the sequence of human CHRAC1, I suggest to look into:https://www.ncbi.nlm.nih.gov/nuccore/NM_017444.6remember the best format to work with sequences is FASTAInfo about pCDNA3.1 can be found at multiple websites.You may access this website to help design primers and explore the sequence:www.molbiotools.com/LEARNING OUTCOMES ASSESSED:1. Be able to describe the major tools used in gene technology and understandhow such tools are used.2. Be able to demonstrate practical competence in key gene manipulationtechniques.3. To have developed a range of key skills including information acquisition fromweb-based and library sources, self-directed learning, critical analysis of data,numeracy, writing and presentation of scientific reports.4. Communicate information effectively in written format.Assessment Criteria. These elements are not necessarily given equal weight in the overall assessment.(Marker: please highlight relevant comments.) ElementsUpper 1stFirstUpper SecondLowersecondThirdFailQuality ofpresentationGreat clarity.Very concise.Entirely logicalin structure.Negligible errorsin spelling &grammar.Clear andconcise.Generally verylogical.Minimal errors inspelling &grammar.Usually clear andconcise. Onlyminorweaknesses inlogic.Few minor errorsin spelling &grammar.Some lack ofclarity and notconcise.Some lapses inlogic. Someerrors inspelling &grammar.Lacks clarity,and notconcise.Many errors inspelling &grammar.Rambling,unclear. Difficultto understand.Very many errorsin spelling &grammar.Quality andrelevance ofinformationComprehensive,accurateinformationcontent. Entirelyrelevant toquestion.All importantinformation.Minimalirrelevance/inaccuracy.Considerableamount ofinformation. Minorirrelevance/inaccuracy.Reasonableamount ofinformation.Someirrelevance/inaccuracy.Limited amountof information.Much irrelevantor inaccurate.Negligibleinformation.Mainlyirrelevant/inaccurate.UnderstandingExcellent.Critical insightand originalityclearly evident.No errors.Very goodunderstanding.Some criticalinsight/originalityshown.Negligible errorsSubstantialunderstanding.Limitedappreciation ofnuances/widerperspectives.Someunderstanding,but rathernarrow.Limited andpatchy.Somewhatmisses thepoint.Little or none.Completelymisses the point.Reading, researchand referencingFull, criticalcoverage ofliterature.Accurately citedand referenced.Broad, criticalcoverage. Onlyminor errors incitation /referencing.Good but lackscritical insight.Generallyaccurate citation /referencing.Adequate butuncritical.Some errors incitation /referencing.Narrow.Uncritical.Numerouserrors incitation /referencing.Little or none.Uncritical.Citation /referencingabsent or full oferrors. FEEDBACK: ADDITIONAL COMMENTS:(Good points/areas for improvement) General comments:Three specific areas for improvement:1. 2.3. Mark: NAME OF MARKER:

QUALITY: 100% ORIGINAL PAPER – NO PLAGIARISM – CUSTOM PAPER

Leave a Reply

Your email address will not be published. Required fields are marked *