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

1
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)

2
Example outcome of a successful transformation
chemically competent cells

1. No DNA--No colonies
2. 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 colonies
4. Vector plus insert--larger number of colonies
   than for 3

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

4

• 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

5
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

6
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

7
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

8
lambda genome
9
Lambda lytic infection
Linear DNA
decision
10
Lambda latent infection (lysogeny)
Lysogen an E. coli strain that can be made to
lyse under the right conditions (e.g. UV
treatment)
11
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)

12
Cloning in lambda phage--an overview
Right arm
Stuffer
Left 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

13
Examples of replacement lambda vectors
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The packaged phage particles are infectious
How to transfer recombinant lambda into cells?
15
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

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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 dont have spi
  gene) can make plaques on lawn of E. coli
  containing a P2 lysogen

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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)

18
M13 structure
Used in phage display techniques
19
ss
ds
Isolate for cloning
M13 life cycle an overview
ss
20
M13 life cycle
Cell has to have F plasmid for infection to work
21
Isolate double-stranded DNA by standard plasmid
prep
M13life cycle
Isolate phage (and single-stranded DNA) in
supernatant
22
M13 doesnt lyse cells, but it does slow them down
lawn of E. coli
M13 infections form plaques, but they are turbid
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M13 mp18 engineered for alpha complementation
24
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.

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• 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.

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Specialized vectors for E.coli

1. Expression vectors
2. Large DNA molecules Cosmids, PACs, and BACs

Course packet 25, 26, 27
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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
28
Expression vectors RNA expression occurs in
vitro (purified plasmids)
29
Making micro RNAs for RNAi one example
30
(No Transcript)
31
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

32
pET vectors protein expression
33
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

34
high affinity, high specificity
Using tags in protein purification
35
A protein purification scheme--removable tag
36
Cloning large DNA fragments

• Cosmids bacteriophage lambda-based
• Bacteriophage P1 plasmids
• BACs F plasmid-based

replicon
transfer
Lambda colE1 P1 P1 F ARS
transfection transfection transfection electropora
tion electroporation transformation
This is a very good table to be familiar with
37
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
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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
39
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

40
Cloning in a cosmid
Desired ligation Products--these are packaged
41
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)
42
Cosmids a specific cloning scheme
Sau3A GATC 5 overhang (compatible with BamHI
sticky end)
split
Prevents multiple fragments
Prevents ligation without insert
43
Phage P1 vectors cloning up to 100 kb DNA
fragments
85-100 kb
44
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
45
Phage P1 vectors cloning up to 100 kb DNA
fragments
PACs like P1 vectors but the DNA is not packaged
(transfer by electroporation)
46
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)

47
General BAC vector
Cloning, etc
selection
7 kb
replication
48
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)
49
Overview of strategies for cloning genes
Get DNA Ligate to vector Transform or
transfect Look for the gene
1)
2)
3)
4)
50
1) Get DNA
RNA
Genomic DNA
51
Ligate to vector how to make this reaction
favorable?
52
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
53
Overview of strategies for cloning genes