1. 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)

2. 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

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 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
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

13. Examples of “replacement” lambda vectors

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

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

16. 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

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

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

23. 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.

25. 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.

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

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

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

29. Making micro RNAs for RNAi: one example

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. Using tags in protein purification
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 F
ARS
transfection
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

38. 99% probability of having a given DNA sequence
17 kb fragment library
Mammalian genome: 3 x 109 base pairs
ln( )
N =
ln( )
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 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

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
Genomic DNA
RNA

51. Ligate to vector: how to make this reaction favorable?

52. 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…

53. Overview of strategies for cloning genes