Design. Construction. Characterization

Transcription

1 Design Construction Characterization

2 DNA mrna (messenger) A C C transcription translation C A C protein His A T G C T A C G

3 Plasmids replicon copy number incompatibility selection marker origin of replication / replicon transcription terminator selection marker Expression Plasmid multi-cloning site promoter transcription terminator your gene of interest promoter

4 Plasmids vs. Chromosomal Insertions

5 De Novo DNA Synthesis 5 end HOCH 2 B O O DNA has directionality: 5 GATC 3 is different than 3 GATC 5 O - P O CH 2 B O O DNA is chemically synthesized 3 to 5 DNA is biologically synthesized 5 to 3 3 end De novo DNA synthesis is currently limited to ~200 nt Due to error rates in synthesis must purify over ~70 nt For de novo synthesis over 200 nt, must use advanced strategies such as assembly

6 PCR primers template denature (~94 C) anneal (~45-60 C) elongate (~72 C) 20-35x denature (~94 C) anneal (~45-60 C) elongate (~72 C) PCR requires: template, primers, thermostable polymerase polymerases vary with fidelity, editing activity, speed

7 Primer Design Primers should have Tms between C Primer sets should have similar Tms Primer sequences flank the region to be amplified, this may limit primer sequence space Primer sequences should be designed to minimize (i) homodimerization, (ii) heterodimerization, (iii) hairpin formation (i) (ii) (iii) Primer design should take into account non-specific binding to other regions on the DNA template (longer is not always better)

8 Modifications to PCR 1. Addition of new sequences within primers 2. PCR assembly PCR 3. Overlap extension PCR PCR break into overlapping primers

9 Ligation reactions 5 P 3 OH vector 3 OH 5 P 5 P 3 OH 3 OH 5 P Intermolecular reaction (effective at high concentrations) Intramolecular reaction (effective at lower concentrations) 1 of many possible products linear, circular, homo and heterodimers insert

10 Cloning 5 protruding ends (EcoRI) G A A T T C C T T A A G 3 protruding ends (PstI) C T G C A G G A C G T C G C T T A A C T G C A G A A T T C G G A C G T C

11 Cloning Non-directional cloning (single restriction site) EcoRI + EcoRI EcoRI digest ligate EcoRI or EcoRI + EcoRI EcoRI

12 Cloning Directional cloning (two non-compatible restriction sites) SalI EcoRI + SalI EcoRI digest ligate SalI EcoRI Blunt end cloning +

13 Klenow fragment used to fill in 5 protruding ends G C T T A A A A T T C G Exonuclease I removes single-stranded nts in 3 -> 5 C T G C A G G A C G T C Phosphatase removes 5 phosphate on a DNA strand G C T T A A A A T T C G

14 Transformations Chemical methods ice cold salt solutions, heat shock; efficiencies ~ colonies/ug scdna Physical methods ice cold salt-free solutions, electrical charge; efficiencies ~ col/ug scdna

15 Screening Restriction mapping SacI 1700 bp 2000 bp 4000 bp EcoRI SalI 1500 bp BamHI Colony PCR Sequencing Confirm sequence of cloned fragment L E/S/S B BamHI 1200 bp Confirm change in PCR fragment in cloned construct distance btwn end of primer and start of desired sequence ~50-70 nt

16 Altering DNA Sequences Rational, targeted alteration through site-directed mutagenesis Small changes to a DNA sequence (~1-5 nt) 1. Design complementary primers with altered DNA sequence (can be specific sequence or randomized sequence) 2. Perform PCR on entire plasmid 3. Digest parent plasmid with restriction enzyme that targets methylated DNA 4. Transform into appropriate host Can perform on fragment and follow with cloning

17 Directed evolution Random mutagenesis Perform PCR under conditions that increase the mutation rate of the polymerase Recombination family of related sequences * * * * * * * * * * * * * * - random digestion of fragments and assembly - directed crossover events and assembly Pulling out functional members of large libraries - selection: an assay that links the desired property of the molecule to cell growth - screen: an assay that links the desired property of the molecule to a detectable output signal

18 Characterization Tools transcription translation DNA mrna protein decay A B Purification of cellular constituents - DNA - RNA -proteins - metabolites

19 mrna Northern blot analysis run RNA out on gel transfer RNA to membrane Real-time quantitative PCR (qrt-pcr) hybridize with labeled probe for transcript of interest detect and quantify the probe signal mrna cdna * * * make cdna copies of the RNA amplify region of the cdna quantify amplification during PCR Differences in relative starting amounts of cdna template are reflected in differences in cycle numbers when exponential phase of amplification is observed signal cycle number

20 mrna Microarrays mrna make cdna copies of total RNA fluorescently label cdnas * cdna hybridize to a chip with complementary ssdna spotted in arrays Microarray methods have been adapted to detecting proteins and metabolites

21 Proteins Western blot analysis run protein out on gel Analytical techniques transfer protein to membrane hybridize with labeled probe to protein of interest (antibody) detect and quantify the probe with a secondary antibody Commonly used liquid chromatography-mass spectrometry (LC-MS), tandem MS-MS, 2-dimensional gel electrophoresis (2D-GE) MS is used for identification LC, gas chromatography, GE, and capillary electrophoresis are used to separate and purify

22 Activity and reporter assays Reporter enzymes Most commonly used is β-galactosidase (encoded by the lacz gene) Plate-based assay Solution / quantitative assay Other commonly used reporter enzymes are luciferases + yellow

23 Activity and reporter assays Reporter proteins Fluorescent reporter proteins are commonly used and require no substrate GFP was original fluorescent reporter protein Researchers have spent significant effort generating: color-shifted variants, more rapidly folding and degrading variants, monomeric forms, brighter fluorescence, resistant to photobleaching, optimized for different organisms FRET applications

24 Activity and reporter assays Use of fluorescent reporter proteins as readouts of cellular components and networks Promoter fusion P GFP Protein fusion P GFP Operon fusion P GFP