Study and engineering of gene function: mutagenesis

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فهرست عناوین اصلی در این پاورپوینت

● Study and engineering of gene function: mutagenesis
● Uses for mutagenesis
● Protein engineering-Why?
● Random mutagenesis
● Cassette mutagenesis (semi-random)
● Random and semi-random mutagenesis: directed evolution
● Gene shuffling
● Screening by phage display: create library of mutant proteins fused to M13 gene III
● Phage display:production of recombinant phage
● Phage display: collect tight-binding phage
● Animation of phage display
● Site-directed mutagenesis: primer extension method
● Alternative primer extension mutagenesis techniques
● “QuikChangeTM” protocol
● Site-directed mutagenesis: Mega-primer method
● PCR-mediated deletion mutagenesis
● Directed mutagenesis
● An example of directed mutagenesis
● The genetic code
● Altering the genetic code
● Why add new amino acids to proteins?
● How to modify genetic code?
● tRNA charging and usage
● Outcome
● Utility of strategy
● Some questions:

نوع زبان: انگلیسی حجم: 1.21 مگا بایت
نوع فایل: اسلاید پاورپوینت تعداد اسلایدها: 38 صفحه
سطح مطلب: نامشخص پسوند فایل: ppt
گروه موضوعی: زمان استخراج مطلب: 2019/06/07 10:54:07

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عبارات مهم استفاده شده در این مطلب

عبارات مهم استفاده شده در این مطلب

mutagenesis, protein, amino, acid, gene, new, pcr, add, random, code, trna, codon,

توجه: این مطلب در تاریخ 2019/06/07 10:54:07 به صورت خودکار از فضای وب آشکار توسط موتور جستجوی پاورپوینت جمع آوری شده است و در صورت اعلام عدم رضایت تهیه کننده ی آن، طبق قوانین سایت از روی وب گاه حذف خواهد شد. این مطلب از وب سایت زیر استخراج شده است و مسئولیت انتشار آن با منبع اصلی است.

http://web.pdx.edu/~justc/courses/RecombDNA/Week7.ppt

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عبارات پرتکرار و مهم در این اسلاید عبارتند از: mutagenesis, protein, amino, acid, gene, new, pcr, add, random, code, trna, codon,

مشاهده محتوای متنیِ این اسلاید ppt

مشاهده محتوای متنیِ این اسلاید ppt

study and engineering of gene function mutagenesis why mutagenize random mutagenesis mutant selection schemes site directed mutagenesis deletion mutagenesis engineering of proteins alterations in the genetic code course packet ۳ uses for mutagenesis define the role of a gene are phenotypes altered by mutations determine functionally important regions of a gene in vivo or in vitro improve or change the function of a gene product investigate functions of non genes eg. dna regions important for regulation protein engineering why enhance stability function under new conditions temperature ph organic aqueous solvent salt alter enzyme substrate specificity enhance enzymatic rate alter epitope binding properties enzymes biotech cash crops from koeller and wang enzymes for chemical synthesis nature ۴ ۹ ۲۳۲ ۲۴ ۲ ۱ obtaining useful enzymes random mutagenesis cassette mutagenesis with doped oligos chemical mutagenesis expose short piece of dna to mutagen make library of clones test for phenotypes pcr mutagenesis by base misincorporation include mn۲ in reaction reduce concentration of one dntp random mutagenesis by pcr the green fluorescent protein screen mutants cassette mutagenesis semi random strands synthesized individually then annealed allows random insertion of any amino acid at defined positions translation of sequence random and semi random mutagenesis directed evolution mutagenize existing protein eg. error prone pcr doped oligo cassette mutagenesis and or do gene shuffling creates library screen library of mutations for proteins with altered properties standard screening ۱ ۱ mutants phage display ۱ ۹ mutants gene shuffling sexual pcr gene shuffling for gene shuffling protocols you must have related genes in original pool ۱ evolutionary variants or ۲ variants mutated in vitro shuffling allows rapid scanning through sequence space faster than doing multiple rounds of random mutagenesis and screening shuffling of one gene mutagenized in two ways gene shuffling cephalosporinase from ۴ bacteria single gene mutagenesis multiple gene shuffling screening by phage display create library of mutant proteins fused to m۱۳ gene iii human growth hormone want to generate variants that bind to hgh receptor more tightly random mutagenesis phage display production of recombinant phage the display phage display collect tight binding phage the selection animation of phage display http www.dyax.com discovery phagedisplay.html site directed mutagenesis primer extension method drawbacks both mutant and wild type versions of the gene are made following transfection lots of screening required or tricks required to prevent replication of wild type strand requires single stranded circular template dna alternative primer extension mutagenesis techniques quikchangetm protocol advantage can use plasmid double stranded dna destroys the template dna dna has to come from dam host site directed mutagenesis mega primer method megaprimer needs to be purified prior to pcr ۲ allows placement of mutation anywhere in a piece of dna a b wild type template first pcr second pcr domain swapping using megaprimers overlapping pcr n c mega primer pcr ۱ pcr ۲ domains have been swapped template ۱ template ۲ pcr mediated deletion mutagenesis target dna pcr products oligonucleotide design allows precision in deletion positions directed mutagenesis make changes in amino acid sequence based on rational decisions structure known mutate amino acids in any part of protein thought to influence activity stability solubility etc. protein with multiple family members mutate desired protein in positions that bring it closer to another family member with desired properties an example of directed mutagenesis t۴ lysozyme structure known can it be made more stable by the addition of pairs of cysteine residues allowing disulfide bridges to form without altering activity of the protein t۴ lysozyme a model for stability studies cysteines were added to areas of the protein in close proximity disulfide bridges could form more disulfides greater stabilization at high t bottom of bar melting temperature under reducing condtions top of bar melting temperature under oxidizing conditions green bars if the effects of individual s s bonds were added together stability can be increased but there can be a cost in activity the genetic code ۶۱ sense codons ۳ non sense stop codons ۲ amino acids other amino acids some in the cell as precursors to other amino acids but very rarely have any been added to the genetic code in a living system is it possible to add new amino acids to the code yes …sort of wang et al. ۲ ۱ expanding the genetic code science ۲۹۲ p. ۴۹۸. altering the genetic code why add new amino acids to proteins new amino acid new functional group alter or enhance protein function rational design chemically modify protein following synthesis chemical derivitization probe protein structure function modify protein in vivo add labels and monitor protein localization movement dynamics in living cells how to modify genetic code adding new amino acids to the code must bypass the fidelity mechanisms that have evolved to prevent this from occurring ۲ key mechanisms of fidelity correct amino acid inserted by ribosome through interactions between trna anti codon and mrna codon of the mrna in the ribosome specific trna charged with correct amino acid because of high specificity of trna synthetase interaction add new trna add new trna synthetase trna charging and usage charging trna amino acid amino acyl trna synthetase translation trna aa codon anticodon interaction ribosome chose trnatyr and the trnatyr synthetase mtyrrs from an archaean m.jannaschii no cross reactivity with e. coli trnatyr and synthetase mutate m trnatyr to recognize stop codon uag on mrna mutate m tyrrs at ۵ positions near the tyrosine binding site by doped oligonucleotide random mutagenesis obtain mutants that can insert o methyl l tyrosine at any uag codon outcome strategy allows site specific insertion of new amino acid just design protein to have uag stop codon where you’d like the new amino acid to go transform engineered e. coli with plasmid containing the engineered gene feed cells o methyl tyrosine to get synthesis of full length gene utility of strategy several new amino acids have been added to the e. coli code in this way including phenyalanine derivatives with keto groups which can be modified by hydrazide containing fluorescent dyes in vivo useful for tracking protein localization movement and dynamics in the cell p acetyl l phenylalanine m acetyl l phenylalanine some questions what are the consequences for the cell with an expanded code do new amino acids confer any kind of evolutionary advantage to organisms that have them assuming they get a ready supply of the new amino acid… why do cells have need ۳ stop codons …

کلمات کلیدی پرکاربرد در این اسلاید پاورپوینت: mutagenesis, protein, amino, acid, gene, new, pcr, add, random, code, trna, codon,

این فایل پاورپوینت شامل 38 اسلاید و به زبان انگلیسی و حجم آن 1.21 مگا بایت است. نوع قالب فایل ppt بوده که با این لینک قابل دانلود است. این مطلب برگرفته از سایت زیر است و مسئولیت انتشار آن با منبع اصلی می باشد که در تاریخ 2019/06/07 10:54:07 استخراج شده است.

http://web.pdx.edu/~justc/courses/RecombDNA/Week7.ppt

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