Setting up a transformation--how will the competent cells be

treated?1. No plasmid (negative control, nothing

should grow on this plate)2. Supercoiled plasmid of a known

concentration (to determine efficiency of competent cells, in transformants/microgram)

3. Vector DNA (dephosphorylated?) ligated without insert DNA (background transformants)

4. Vector DNA ligated with insert DNA (desired products)

Example outcome of a successful transformation: chemically competent

cells

1) No DNA--No colonies2) 2 nanograms (10-9 g, 10-3 micrograms)

supercoiled plasmid DNA--500 colonies (efficiency of cells: 2.5 x 105 transformants per microgram DNA)

3) Vector alone--small number of colonies4) 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

I. Lambda: a “head and tail” phage

--The lambda life cycle--Basic cloning in lambda

II. M13: a filamentous phage--Life cycle--genome structure--phagemids

Bacteriophages

• Viruses that infect bacteriaa) “head and tail”b) Filamentousc) 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

decision

Linear DNA

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 Right arm“Stuffer”

1) 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?

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

ss

dsIsolate for cloning

M13: life cycleCell has to have F plasmid for infection to work

M13:life cycle

Isolate phage (and single-stranded DNA) in supernatant

Isolate double-stranded DNA by standard plasmid prep

M13 doesn’t lyse cells, but it does slow them down

M13 infections form plaques, but they are “turbid”

“lawn” of E. coli

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

I. Expression vectors

II. 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:

promoter MCS

tags tags

Transcriptionterminator

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

A protein purification scheme--removable tag

Cloning large DNA fragments• Cosmids: bacteriophage lambda-based• Bacteriophage P1 plasmids• BACs: F plasmid-based

replicon transfer

LambdacolE1

P1P1F

ARS

transfectiontransfectiontransfectionelectroporationelectroporationtransformation

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?

N = ln(1-p)

ln(1-f)

P = probability of getting a specific piece of the genome (1.0 = 100%)f = fractional size of clone DNA relative to genome

N = number of clones needed

N = ln(1 - 0.99)

ln(1 - )1.7 x 104

3 x 109

99% probability of having a given DNA sequence

17 kb fragment library

Mammalian genome: 3 x 109 base pairs

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

split

Sau3A: GATC 5’ overhang (compatible with BamHI sticky end)

Prevents ligation without insert

Prevents multiple fragments

Phage P1 vectors: cloning up to 100 kb DNA fragments

85-100 kb

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

replication

selection

Cloning, etc

7 kb

o---- Cloning strategies ----o

I. Making DNA “libraries” (from genomic DNA, mRNA “transcriptome”)

II. 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

1)

2)

3)

4)

Get DNA

Ligate to vector

Transform or transfect

Look for the gene…

Genomic DNA RNA

1) Get DNA

Ligate to vector: how to make this reaction favorable?

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