Setting up a transformation--how will the competent cells be treated? No plasmid (negative control, nothing should grow on this plate) Supercoiled plasmid of a known concentration (to determine efficiency of competent cells, in transformants/microgram) Vector DNA (dephosphorylated?) ligated without insert DNA (background transformants) Vector DNA ligated with insert DNA (desired products)

Example outcome of a successful transformation: chemically competent cells No DNA--No colonies 2 nanograms (10-9 g, 10-3 micrograms) supercoiled plasmid DNA--500 colonies (efficiency of cells: 2.5 x 105 transformants per microgram DNA) Vector alone--small number of colonies Vector plus insert--larger number of colonies than for #3

Identifying recombinant plasmid-containing cells Alpha complementation: most white colonies represent presence of insert DNA blocking functional beta galactosidase Increase in number of transformants in presence of insert vs. absence of insert Insert treated with alkaline phosphatase Directional cloning--preventing religation of vector SCREEN colonies/plasmids for inserts, usually by PCR Confirm clones by sequencing

Mobilizing DNA: vectors for propagation in E. coli Plasmids Bacteriophage M13 Lambda Specialized cloning vectors expression vectors and tags vectors for large pieces of DNA, e.g. Cosmids and BACs

Bacteriophages: useful vectors in molecular cloning Lambda: a “head and tail” phage --The lambda life cycle --Basic cloning in lambda M13: a filamentous phage --Life cycle --genome structure --phagemids

Bacteriophages Viruses that infect bacteria a) “head and tail” b) Filamentous c) etc…. Nucleic acid molecule (usually DNA) Carrying a variety of genes for phage replication Surrounded by a protective protein coat (capsid) Infection (instead of transformation): Phage attaches to outside of bacterium, injects DNA Phage DNA is replicated Capsid proteins synthesized, phage assembled and released

Bacteriophage lambda “head and tail” phage, very well-studied Large, linear genome--48.5 kb Central region of genome (“stuffer”) is dispensable for infectious growth--it can be engineered out Two lifestyle modes Lytic: replicative mode Lysogenic: latent mode Useful for cloning 5-25 kb DNA fragments

lambda genome

Lambda: lytic infection Linear DNA decision

Lambda: latent infection (lysogeny) Lysogen: an E. coli strain that can be made to lyse under the right conditions (e.g. UV treatment)

Lambda as a cloning vector Insertional vectors (clone into single restriction site, can only increase genome size by 5% (size of foreign DNA insert depends on the original size of the phage vector, about 5 to 11 kb) Replacement vectors (removing “stuffer”), can clone larger pieces of DNA, 8 to 24 kb (sufficient for many eukaryotic genes)

Cloning in lambda phage--an overview Left arm “Stuffer” Right arm Restrict, purify right and left arms 2) Ligate with foreign DNA 3) “Package” ligation mixture into phage heads 4) Plate mixture on E. coli, individual plaques represent recombinant clones

Examples of “replacement” lambda vectors

The packaged phage particles are infectious How to transfer recombinant lambda into cells? The packaged phage particles are infectious

Selecting recombinant lambda phages I There is a minimal size of DNA that can be packaged in lambda phage heads Remove stuffer (for some replacement vectors), the ligated “arms” cannot be packaged without an insert present Selection: only thing that is infectious is the recombinant DNA product

Selecting recombinant lambda phages II Wild type lambda cannot grow on E. coli infected with phage P2 (spi, or sensitive to P2 inhibition), spi+ conferred by red and gam genes in “stuffer” Only phage lacking stuffer (they don’t have spi gene) can make plaques on lawn of E. coli containing a P2 lysogen

Filamentous phages: M13 Single-stranded, circular genome, 6.4 kb Can clone pieces of DNA up to 6X the M13 genome size (36 kb) -- but the larger the DNA, the less stable the clone is….. Useful for Sequencing Site-directed mutagenesis (later) Any other technique that requires single stranded DNA Drawback: foreign DNA can be unstable (slows down host cell growth, so deletions confer a selective advantage)

M13 structure Used in ‘phage display’ techniques

M13 life cycle: an overview ss ds Isolate for cloning M13 life cycle: an overview ss

M13: life cycle Cell has to have F plasmid for infection to work

M13: life cycle Isolate double-stranded DNA by standard plasmid prep Isolate phage (and single-stranded DNA) in supernatant

M13 doesn’t lyse cells, but it does slow them down “lawn” of E. coli M13 infections form plaques, but they are “turbid”

M13 mp18: engineered for alpha complementation

Phagemids: plasmid/M13 hybrids Plasmids containing both plasmid (colE1) origin and bacteriophage M13 origin of replication To recover single-stranded version of the plasmid (for sequencing, e.g.), infect transformed (male) strain with a helper phage (M13KO7) Helper phage cannot produce single stranded copies of itself, but provides replication machinery for single-stranded copies of the phagemid DNA Phagemid single stranded DNA is packaged and extruded into supernatant--can then be isolated for sequencing, etc.

Uses of Bacteriophages: Lambda -- large-ish DNA fragments for gene cloning (large eukaryotic genes) Excellent selection capability (stuffer stuff) Clone lots of precisely-sized DNA fragments for library construction M13 -- single-stranded DNA Sequencing Site-directed mutagenesis Etc.

Specialized vectors for E.coli Expression vectors Large DNA molecules: Cosmids, PACs, and BACs Course packet: #25, 26, 27

Expression vectors For production of specific RNA or protein of interest Optimized for transcription, translation, and post-translational handling Typical expression vector cloning site: Transcription terminator MCS promoter tags tags

Expression vectors: RNA: expression occurs in vitro (purified plasmids)

Making micro RNAs for RNAi: one example

How to control transcription driving RNA/protein expression in vivo? T7 RNA polymerase promoters: T7 RNA polymerase under control of lac repressor (induced by IPTG) Lambda PL promoter, controlled by lambda repressor (which is regulated by trp repressor) pBAD promoter, controlled by the araC protein in response to arabinose

pET vectors: protein expression

Helper tags for protein production and purification 6/7 histidine tag: interacts very specifically with Ni2+ ions, which can be immobilized on columns or beads Biotin carboxylase: covalently attaches to biotin, biotin binds to streptavidin which can be immobilized on columns or beads Epitopes (e.g. c-myc) for specific antibodies can be included as tags--purify on antibody column Tags can be engineered to be removable

Using tags in protein purification high affinity, high specificity Using tags in protein purification

A protein purification scheme--removable tag

Cloning large DNA fragments Cosmids: bacteriophage lambda-based Bacteriophage P1 plasmids BACs: F plasmid-based replicon transfer Lambda colE1 P1 F ARS transfection electroporation transformation This is a very good table to be familiar with

Why clone large pieces of DNA??? Make libraries: genome broken up into small, manageable, organizable pieces Each recombinant DNA fragment from the ligation--a piece of the genome How many recombinant DNA molecules are required in a library to get complete coverage of a genome? P = probability of getting a specific piece of the genome (1.0 = 100%) ln(1-p) N = ln(1-f) f = fractional size of clone DNA relative to genome N = number of clones needed

99% probability of having a given DNA sequence 17 kb fragment library Mammalian genome: 3 x 109 base pairs ln(1 - 0.99) N = ln(1 - ) 1.7 x 104 3 x 109 N = 8.1 x 105 clones required

Cosmids: 5 kb plasmids, antibiotic resistance, plasmid origin of replication Contain lambda cos sites required for packaging into lambda phage heads Packaging only occurs with 37-52 kb fragments--selection for large fragments Packaged DNA is inserted into cells and then replicates as a very large plasmid

Cloning in a cosmid Desired ligation Products--these are packaged

Cloning in a cosmid Instead of transformation, desired ligation products are packaged and then transfected into cells Selection for colonies, not screening of plaques (not infectious)

Cosmids: a specific cloning scheme Sau3A: GATC 5’ overhang (compatible with BamHI sticky end) split Prevents multiple fragments Prevents ligation without insert

Phage P1 vectors: cloning up to 100 kb DNA fragments

Phage P1 vectors: cloning up to 100 kb DNA fragments Efficiency of packaging is typically low: thus it is not good for making large genomic libraries

Phage P1 vectors: cloning up to 100 kb DNA fragments PACs: like P1 vectors but the DNA is not packaged (transfer by electroporation)

BACs: Bacterial Artificial Chromosomes Based on the F factor of E. coli: --100 kb plasmid, propagates through conjugation --low copy number (1-2 copies per cell) --2 genes (parA and parB): accurate partitioning during cell division BACs: just have par genes, replication ori, cloning sites, selectable marker Can propagate very large pieces of DNA: up to 300 kb Relatively easy to manipulate: move into cells by transformation (electroporation)

General BAC vector Cloning, etc selection 7 kb replication

o---- Cloning strategies ----o Making DNA “libraries” (from genomic DNA, mRNA “transcriptome”) Screening to identify a specific clone (the needle in the haystack) -- by the sequence of the clone -- by the structure or function of the expressed product of the clone Course reading: #28 (and 29)

Overview of strategies for cloning genes Get DNA Ligate to vector Transform or transfect Look for the gene… 1) 2) 3) 4)

1) Get DNA Genomic DNA RNA

Ligate to vector: how to make this reaction favorable?

Hence “liver” vs. “brain” vs. “heart” cDNA libraries This yields a “library”, a representative set of all the pieces of DNA that make up a genome (or all the cDNAs that correspond to the “transcriptome”) cDNAs from different tissues reflect the different RNA populations that you find in distinct cell types: Hence “liver” vs. “brain” vs. “heart” cDNA libraries There are lots of ways to identify a particular gene…

Overview of strategies for cloning genes