Site-Directed Mutagenesis

01. GFP & GFP Derivatives 02. Site-Directed Mutagenesis 03. pET Expression System

GFP&GFP Derivatives Wild type GFP Green fluorescent protein (GFP) traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. GFP is a protein which exhibits bright green fluorescence when exposed to blue light. The protein composed of 238 amino acids (26.9kDa). The protein sequence and its resultant three-dimensional folding structure into the 11-strand β-barrel, is most likely crucial to the formation of the chromophore and its bioluminscene. http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html

GFP&GFP Derivatives Wild type GFP The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.

Formation of the GFP Chromopore GFP&GFP Derivatives Formation of the GFP Chromopore GFP has the ability to exhibit intrinsic fluorescence thanks to three amino acids that cyclise (Ser65-Tyr66-Gly67) and then undergo an oxidation step during a complex maturation process. http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html

Limitations with Wild-type GFP GFP&GFP Derivatives Limitations with Wild-type GFP Wild-type GFP can be excited by light at two different absorption peaks, which the optimal excitation one is by UV light. This is problematic if one wants to use GFP in live cells, since UV radiation can damage them. Wild-type GFP folds poorly at 37°C, so it limits its usefulness for in vivo experiments in the lab. It can take up to four hours to fold GFP correctly

GFP&GFP Derivatives GFP Derivatives A diversity of mutants have been derived from GFP to emit different wavelengths of light; these include : cyan fluorescent protein (CFP) yellow fluorescent protein (YFP) blue fluorescent protein (BFP)

GFP&GFP Derivatives GFP Derivatives Changes in the amino acids in the fluorophore region, as well as in amino acids downstream from the fluorophore, result in changes in the color the fluorophore emits. http://www.chm.bris.ac.uk/motm/GFP/GFPh.htm

GFP Derivatives GFP&GFP Derivatives http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html

Mutagenesis Methods of mutagenesis Site-Directed Mutagenesis Random DNA recombination Targeted Random change in residues or exhaustive enumeration of amino acid substitutions within selected sites Point mutations

Approaches for directed mutagenesis Site-Directed Mutagenesis Approaches for directed mutagenesis -> Site-directed mutagenesis -> Point mutations in particular known area Result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species -> Random mutagenesis -> Point mutations in all areas within DNA of interest Result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening If methods efficient -> mostly mutated DNA

Point mutations (Directed mutagenesis) Site-Directed Mutagenesis Point mutations (Directed mutagenesis) Point mutations (directed mutagenesis) Substitution: change of one nucleotide (i.e. A-> C) Insertion: gaining one additional nucleotide Deletion: loss of one nucleotide http://en.wikipedia.org/wiki/File:Point_mutations-en.png

Site-Directed mutagenesis PCR-based methods PCR Substitution PCR Deletion Site-directed mutagenesis by traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon. PCR Terminal Substitution

Polymerase Chain Reaction (PCR) Site-Directed Mutagenesis Polymerase Chain Reaction (PCR) An in vitro method for the enzymatic synthesis of specific DNA sequences Requires Two specific oligonucleotide primers DNA polymerase dNTPs Template DNA https://www.neb.com/applications/dna-amplification-and-pcr

Site-Directed mutagenesis http://www.genomics.agilent.com/article.jsp?pageId=388&_requestid=610179

Site-Directed mutagenesis Primer design guidelines The mutagenic oligonucleotide primers for use in this protocol must be designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid. Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78 ℃. The desired mutation (deletion or insertion) should be in the middle of the primers with ~10-15 base of correct sequence on both sides. The primers optically should have a minimum GC content of 40 % and should terminate in one or more C or G bases

Site-Directed mutagenesis Primer design example DNA sequence : AT->GC 5’-agaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg-3’ 3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’ 5’-agaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg-3’ 3' CAGGGTTAAGAACAACT ATCTACCACTACAATTAC 5‘ CG GC 5' GTCCCAATTCTTGTTGA TAGATGGTGATGTTAATG 3' 3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’ GC content: 40.54%         Melting temp: 75.1 °C         Mismatched bases: 2 Length: 37 bp                 Mutation: Substitution 5' flanking region: 17 bp    3' flanking region: 18 bp    

Protein Expression Strategy pET Expression system Protein Expression Strategy

Genetic Elements Essential for Expression pET Expression system Genetic Elements Essential for Expression Expression cassette Promoter Regulator binding site Ribosome binding site (RBS) Multiple cloning site (MCS) Transcription terminator Antibiotic resistance gene Origin of replication Other control gene

pET Expression system pET system The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction.

pET system Promoter & Expression genes pET Expression system pET system Promoter & Expression genes lac promoter (host) – T7 RNA polymerase T7 promoter (vector) – Target gene Regulator binding site (lac O) Control gene (lac I) T7 RNA polymerase inhibitor (T7 lysozyme)

Working Mechanism of pET vector system pET Expression system Working Mechanism of pET vector system http://shiyan.ebioe.com/ExpressionRecombinantProteins_2.htm

Experimental Scheme EGFP → EYFP 2014.04.01 PCR 2014.04.08 Products are checked by electrophoresis 2014.04.01 PCR 2014.04.08 Treatment of Dpn I Making calcium competent cells (E. coli DH5a, E. coli BL21 (De3)) Transformation into E. coli DH5a 2014.04.15 Plasmid preparation Transformation into E. coli BL21 (De3) Expression of EYFP

Report Materials & Methods EGFP와 EYFP nucleotide, protein sequence 찾아서 비교하기 203번 amino acid를 치환하기 위한 Primer design (GC content, Tm, base pair…) PCR method 정리

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