Lectur 9 Slides (Cloning a gene I; a classic approach) (1).pdf

Biol 366: A classic approach to cloning a gene: Cloning of limonene synthase cDNA from spearmint. Learning outcome: 1. How do construct a cDNA library 2. How to produce a probe for a protein-encoding gene for which sequence is not known 3. How to screen a cDNA library to clone a protein-encoding gen...

Biol 366: A classic approach to cloning a gene: Cloning of limonene synthase cDNA from spearmint. Learning outcome: 1. How do construct a cDNA library 2. How to produce a probe for a protein-encoding gene for which sequence is not known 3. How to screen a cDNA library to clone a protein-encoding gene, for which DNA sequence is unknown Text Section: Section 7.1.3 Additional Reading: Reference 1: Alonso WR, Rajaonarivony JI, Gershenzon J, Croteau R. Purification of 4Slimonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata). J Biol Chem. 1992 Apr 15;267(11):7582-7. Reference 2: S M Colby, W R Alonso, E J Katahira, D J McGarvey, R Croteau. 4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. Journal of Biological Chemistry). 12/1993; 268(31):23016-24. ZAP Express cDNA Synthesis Kit Manual (pages 1- 12) Review: DNA RNA So far have discussed: 1. Chemical Synthesis of DNA 2. Amplification of DNA (PCR) 3. Sequencing DNA Protein - Catalytic - Structural - Hormones - Sensors - Receptors - Etc. 4. Engineering and cloning a DNA fragment in a vector Next Topic: Cloning a (protein-encoding) gene. Good example: Assignments Melanie Lemaire discussing using cDNA libraries to clone new hazelnut allergens not found in pollen. 2 Why clone a gene? - Understanding gene function - Modulating gene expression to: - Produce r-proteins in heterologous systems - Enhance biological processes in vitro - Enhance biological processes in vivo - Introduce novel traits in organsms - Etc. 3 Unmarked in-class activity. What is the DNA sequence that encodes the following peptide? M-A-C-H 4 * * What is the DNA sequence that encodes the following peptide? M-A-C-H (Met-Aal-Cys-His) * Met-Aal-Cys-His ATG-GCA-TGC-CAC ***-GCC-TGT-CAT ***-GCG-***-*** ***-GCT-***-*** * = same as above nucleotide 5 Utility of cDNA libraries in cloning genes Several approaches are possible. We will look at two approaches. Approach 1: A classical strategy. • Produce a cDNA library that includes the target gene • Obtain a probe for the gene of interest • Screen the library with the probe • Isolate a full length clone • Express the clone in bacteria Fig 1: Colby et al., 1999. • Purify the recombinant-protein and establish function Example: Cloning of limonene synthase (LimS) cDNA from spearmint. 6 Cloning a gene from a cDNA library A) Building a cDNA library 1) Obtain total RNA / mRNA (preferred) 2) Ligate Poly T (why poly T????) 3) Use RT enzyme to produce cDNA 7 Building a cDNA library (Section 7.1.3) 4) Degrade the mRNA 5) Add an adaptor / linker (of known sequence) 6) Add a forward primer and produce ds-cDNA 7) Clone the gene of interest • Use cDNA library directly (by PCR) to amplify & clone a gene • Clone each cDNA in a vector, and transform into bacterial cells (see next side) 8 Cloning a gene (cDNA) that encodes a protein of interest? Workflow: -Build a cDNA library -Find a probe -Screen the library with the probe to isolate the target cDNA (red) Note: A cDNA library is a pool of cDNAs, each of which has been cloned in a vector 9 Cloning a gene from a cDNA library Screening the cDNA library using the probe . . . . . . . . . . . . . .. . . . 1. Grow bacteria containing cDNA 2. Transfer plasmid DNA from bacterial to a nitrocellulose membrane. . . . . . . . . . . . . . . . . . . 3. Screen the membrane with a probe specific for the gene of interest (Southern blotting) 10 Building a cDNA library (more notes) Fig 1: ZAP Express cDNA Synthesis Kit Manual (cDNA synthesis flow chart) 11 Building a cDNA library, continued Fig 1: ZAP Express cDNA Synthesis Kit Manual (cDNA synthesis flow chart) Why directional cloning (using EcoR I/Xho I)???? 12 Building a cDNA library, continued. • Clone all cDNA species into XhoI / EcoRI sites of pBK-CMV vector • Transform construct into E. coli calls (one construct per cell) Why use EcoRI / XhoI, instead of just EcoRI????? XhoI . . . EcoRI Fig 3: ZAP Express cDNA Library Manual 13 Building a cDNA library (some notes) • Some genes can be underrepresented: – Genes expressed at low level – Genes for which mRNA is not stable • cDNA libraries can be normalized to enrich for unstable or poorly expressed mRNA using various methods. 14 In class activity - ungraded Why is methylated cDNA made in lambda Zap cDNA synthesis kits? 15 Cloning a gene from a cDNA library Case study: Cloning the cDNA that encodes the limonene synthase (LimS) enzyme from a mint cDNA library. LimS Fig 1: Colby et al., 1999. 16 Case study: Cloning limonene synthase (LimS) cDNA In this study, the researchers: • Obtained a DNA probe for LimS • Built a spearmint cDNA library • Screened a spearmint cDNA library using the LimS probe • Isolated LimS cDNA • Expressed the cDNA in bacteria to obtain r-protein • Assayed the r-protein to confirm function Reference: S M Colby, W R Alonso, E J Katahira, D J McGarvey, R Croteau (1993). 17 To produce a DNA probe for an “unknown” protein-coding gene: • Obtain purified protein from the target tissue using chromatography • Break up the protein into small pieces • Obtain amino acid sequence for a few fragments • Deduce DNA (coding) sequence for protein fragments • Design PCR primers against the deduced cDNA • Extract RNA from target tissue, and reverse transcribe to cDNA • Using primers amplify a fragment of the target cDNA using PCR Reference: S M Colby, W R Alonso, E J Katahira, D J McGarvey, R Croteau (1993). 18 Cloning of limonene synthase: Obtaining a probe Step 1: Purification of the protein The 4S-limonene synthase protein (enzyme) was purified using THREE chromatography steps. • Dye ligand chromatography • Anion exchange chromatography • Hydrophobic interaction chromatography – This is time consuming and tedious – Requires expertise and equipment Figure 4. SDS PAGE of purified LinS (Alanso et al., 1992) Reference: S M Colby, W R Alonso, E J Katahira, D J McGarvey, R Croteau (1993). 19 19 Step 2. Obtaining a probe for screening a cDNA library Reference 2: S M Colby, W R Alonso, E J Katahira, D J McGarvey, R Croteau. 4S-limonene synthase from the oil glands of spearmint (Mentha spicata). cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. Journal of Biological Chemistry). 12/1993; 268(31):23016-24. .Internal amino acid sequences of the purified enzyme from spearmint oil glands were utilized to design three distinct oligonucleotide probes. These probes were subsequently employed to screen a spearmint leaf cDNA library, and four clones were isolated. ………… Abstract: ……………………. 20 Cloning a gene from a cDNA library. Obtaining a probe • The purified LimS protein (below) was fragmented using Cyanogen Bromide • Amino acid sequence for each piece was determined. How?????? • The cDNA coding the fragments were deduced to design degenerate primers • Degenerate primers were designed based on amino acid sequence • A probe was amplified by PCR and used to screen a cDNA library Taken from Fig 2. in Colby et al, 1993. Figure 4. Alanso et al., 1992 21 Cloning a gene from a cDNA library. Obtaining a probe Using forward and reverse primers several DNA fragments (probes) were amplified by PCR from the cDNA library. Green arrow: Forward primer Red arrow: Reverse primer Amino acid sequence for part of the LimS protein. Modified from Fig 4 in Colby et al., 1993 22 Cloning a gene from a cDNA library Step 3: The probes were used to screen a mint cDNA library Screening yielded several colonies containing the LimS cDNA . . . . . . . . . . . . . .. . . . 23 Cloning a gene from a cDNA library 4. Grew positive colonies. 5. Extracted plasmid DNA from each colony 6. Sequenced the gene inserted into the plasmid. What platform???? 7. Expressed cloned cDNA in bacterial cells & produced r-protein 8. Confirmed activity of the encoded protein - How? 24 limonene • The LimS cDNA was expressed in bacterial to produce recombinant LimS • The r-LimS produced limonene from GPP in vitro (GPP) Modified from Fig. 3 in Colby et al., 1993. Detection of monoterpenes produced by the LimS by radio radio gas-liquid chromatography. 25 Activity / Take home assignment (10 points). Which of the following primers can be used to amplify the cDNA encoding the polypeptide “FFM(X92)WMC”? Note: “X92“ stands for 92 unknown amino acids. A) Forward: TT(T/C)TT(C/T)ATG & Reverse: TGGATGTG(C/T) B) Forward: TGGATGTG(C/T) & Reverse: TT(T/C)TT(C/T)ATG C) Forward: TT(T/C)TT(C/T)ATG & Reverse: ACCTACTC(G/A) D) Forward: TT(T/C)TT(C/T)ATG & Reverse: (A/G)CA CAT CCA Show your work. 26 In class activity (ungraded) Which of the following primers can be used to amplify the cDNA encoding the polypeptide “FFM(X92)WMC”? Note: “X92“ stands for 92 unknown amino acids. TT(T/C)TT(C/T)ATG(X92)TGG ATG TG(T/C) ---------------------ACC TAC AC(A/G)5’ A) Forward: TT(T/C)TT(C/T)ATG & Reverse: TGGATGTG(C/T) B) Forward: TGGATGTG(C/T) & Reverse: TT(T/C)TT(C/T)ATG C) Forward: TT(T/C)TT(C/T)ATG & Reverse: ACCTACTC(G/A) D) Forward: TT(T/C)TT(C/T)ATG & Reverse: (A/G)CA CAT CCA Show how the primers will bind the template. 27