E.R. Gauthier, Ph.D.CHMI 4226 F1 Recombinant DNA Technology CHMI 4226 E Week 2 12 January 2009 Toolbox part 2. Plasmids, DNA cloning 101, DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F2 Plasmids Plasmids: –Extrachromosomal (episomal) circular DNA molecules –Minimal features: Origin of replication: allow the plasmid to replicate autonomously Antibiotic resistance gene: allow for selection for bacterial cells that have taken up the vector Multiple cloning site (MCS): a small region of the plasmid engineered to contain the cleavage sites for a limited number of restriction enzymes

E.R. Gauthier, Ph.D.CHMI 4226 F3 Plasmids – origin of replication Origin of replication (Ori): small DNA sequences allowing the bacterial DNA polymerase to bind and initiate DNA replication; Several Ori can be found. ColE1 is the most frequently encountered; Some Ori allow the plasmid to replicate frequently (high copy number plasmids – up to 100 copies per cell); others allow only a low rate of replication initiation (low copy number plasmids – only a few copies per cell)

E.R. Gauthier, Ph.D.CHMI 4226 F4 Plasmids – selection genes Genes which encode for proteins affording resistance to a specific antiibotic; Most frequently encountered: –Amp r : resistance to ampicillin –Kan r : resistance to kanamycin –Tet r : resistance to tetracyclin Bacteria possessing a plasmid with the Amp r gene will survive when plated onto a media containing ampicillin;

E.R. Gauthier, Ph.D.CHMI 4226 F5 Plasmids – multiple cloning sites MCS: a limited region on the plasmid which has been engineered to contain unique cleavage sites for a selected number of restriction enzymes; RE cleavage sites in the MCS are not found anywhere else on the plasmid.

E.R. Gauthier, Ph.D.CHMI 4226 F6 Plasmids – other features Promoters for viral RNA polymerases Sp6, T7 or T3 –Allows for in-vitro trasncription experiments Origin of replication of phage F1: –Enables the production of single-stranded plasmids Genes encoding supressor tRNAs Reporter genes: –Allows for the easy identification of bacterial cells with desired features –E.g.: Lac z (encodes  -galactosidase)

E.R. Gauthier, Ph.D.CHMI 4226 F7 Plasmids – SupF selection gene SupF: –Suppressor tRNA –Inserts an amino acid (Glu) at the UAA stop codon –Allows the survival of bacteria with a P3 episome : a plasmid containing the Amp r and Tet r genes both interrupted with a UAA stop codon.

E.R. Gauthier, Ph.D.CHMI 4226 F8

E.R. Gauthier, Ph.D.CHMI 4226 F9 Plasmids – pBR322

E.R. Gauthier, Ph.D.CHMI 4226 F10 Plasmids - pBluescript

E.R. Gauthier, Ph.D.CHMI 4226 F11 Plasmids – shuttle vectors Shuttle vectors can be used in at least two different organisms: –Bacteria (mandatory) –Yeast –Insects –plants –mammals

E.R. Gauthier, Ph.D.CHMI 4226 F12 Plasmids – shuttle vectors Bacteria/Yeast shuttle vector; Leu2: –Selection marker for growth in yeast –Encodes for a gene which allows for the synthesis of the amino acid leucine in a yeast strain which cannot produce Leu on its own (auxotrophic) –Yeast with this vector can be grown on media devoid fo leucine.

E.R. Gauthier, Ph.D.CHMI 4226 F13 DNA cloning Why cloning DNA? –To produce greater amounts of YFG –To characterise the properties of YFG sequencing Mutagenesis –To express YFP in vitro or in a living organism production of recombinant insulin: much better than insulin purified from blood, which can be contaminated with viruses (hepatitis C, HIV) or other nice things (prions)

E.R. Gauthier, Ph.D.CHMI 4226 F14 DNA cloning – basic steps 1. cut DNA of interest and plasmid with appropriate restriction enzyme 2. mix together and seal free ends with DNA ligase (ligation) 3. Insert DNA in bacteria (transformation) –Requires transformation-competant bacteria 4. Select antibiotic-resistant bacteria and search for cells having the recombinant plasmid. 5. Confirm cloning –restriction enzyme digest –DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F15 DNA cloning – basic steps

E.R. Gauthier, Ph.D.CHMI 4226 F16 DNA cloning Treating vector with alcaline phosphatase –reduces background of bacteria with plasmid not containing the insert. Blunt-ends can be used: –a greater insert DNA/plasmid ratio must be used: 10/1 instead of the usual 3/1 –Much, much less efficient than cloning with protruding ends The ends of your DNA fragment can be modified with Klenow enzyme to accomodate the available RE sites in the vector (and vice-versa). Adaptors and linkers can be used to facilitate cloning.

E.R. Gauthier, Ph.D.CHMI 4226 F17 Adaptors and linkers Adaptors Linkers

E.R. Gauthier, Ph.D.CHMI 4226 F18 Assignment #2

E.R. Gauthier, Ph.D.CHMI 4226 F19 Use of  -gal - Bacteria used in genetic engineering express a portion of  -gal missing the N- terminal 50 amino acids (called the omega [  ] fragment); -Several vectors carry the missing N- terminal 50 amino acids (  fragment) of  - gal in the MCS; - Only the bacteria having both the  and  portion of  -gal can express a functional enzyme (  complementation)

E.R. Gauthier, Ph.D.CHMI 4226 F20 Use of  -gal If a DNA fragment is inserted into the MCS of pBluescript, it interrupts the  -gal  fragment, making it inactive; SO: bacteria with a recombinant plasmid will not express a functional  -gal, and will turn white upon staining wity X-gal; However, bacteria with a plasmid which does not contain the DNA fragment will produce the  fragment, will have a functional  -gal enzyme, and will turn blue upon staining with X-gal.

E.R. Gauthier, Ph.D.CHMI 4226 F21 DNA cloning – plasmid purification from bacteria Culture 1 bacterial colony into liquid media Recover bacterial cells by centrifugation 1) Resuspend cells in glucose- based buffer 2) Lyse bacteria with SDS/NaOH buffer 3) Precipitate bacterial chromosome and proteins with acidic buffer and centrifuge Extract supernatant with basic (pH 8) phenol - solubilizes contaminating proteins Extract with chloroform – to get rid of the phenol Precipitate plasmid DNA with ethanol Dry pellet, resuspend in buffer, digest with restriction enzyme and analyse on agarose gel

E.R. Gauthier, Ph.D.CHMI 4226 F22 DNA cloning – identification of recombinant plasmids Three important tests have to be done: –Cut with RE to see if the insert is of the correct size; –Cut with RE to confirm the identity of the fragment (usually choose RE that cut inside the fragment) –Sequence the insert.

E.R. Gauthier, Ph.D.CHMI 4226 F23 DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F24 DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F25 DNA sequencing 3’ A G T C G C A C T A G T G C A T A G 5’

E.R. Gauthier, Ph.D.CHMI 4226 F26 DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F27 DNA sequencing

E.R. Gauthier, Ph.D.CHMI 4226 F28 What to do with the data? 1. identification (did I clone the piece of DNA I wanted) 2. Is it complete, or do did I clone only a portion of it (happens more often than you think…)? Are there any mutations (happens a lot…)?

E.R. Gauthier, Ph.D.CHMI 4226 F29 BLAST search Blast: algorithm that allows you to quickly identify sequences that are similar to the DNA of interest; Blastp: similarity searches with amino acid sequences Blastn: similarity searches with nucleotide sequences

E.R. Gauthier, Ph.D.CHMI 4226 F30 BLAST search QUERY sequence(s) BLAST database BLAST program BLAST results

E.R. Gauthier, Ph.D.CHMI 4226 F31 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F32 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F33 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F34 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F35 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F36 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F37 BLAST search E value: indicate the odds that the hit is attributable to chance only. –The lower the E value, the better the odds that it is a real match. G: link to Entrez Gene U: link to sequence- related info (expression profile, chromosomal location, orthologs) E: link to GEO (Gene Expression Omnibus): database of gene expression profiles.

E.R. Gauthier, Ph.D.CHMI 4226 F38 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F39 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F40 Translation of sequencing data Allows you you determine if the sequence you isolated contains the entire open reading frame. –Open reading frame (ORF): the nucleotide sequence coding for a protein; Starts with a AUG codon, and stops with one of the 3 stop codons (UAA, UAG, UGA).

E.R. Gauthier, Ph.D.CHMI 4226 F41 BLAST search

E.R. Gauthier, Ph.D.CHMI 4226 F42 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F43 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F44 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F45 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F46 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F47 Translation of sequencing data

E.R. Gauthier, Ph.D.CHMI 4226 F48 Blastp result

E.R. Gauthier, Ph.D.CHMI 4226 F49 Assignment #3!