1. Lecture 6
January 12, 2016
Biotech 3

2. Lecture Topics Cloning Vectors Plasmids Bacteriophage Cosmids
BACS and YACs
3. PCR
General overview and History: 1983 Kary Mullis
Mathematical concept and cycle number
Polymerase rate and fidelity
Forward and Reverse Primer design
Affinity tags Tm
Secondary structure
Primer design for point mutations
PCR Parameters: DNA sequence, primers, type of polymerase, melting temperature, extension time, number of cycles
2. Plasmids
Origin of Replication
Selectivity Markers
Multiple Cloning Site
Promoter Sequence

3. A. Cloning Vectors
Cloning vector: small piece of DNA that can be stably maintained in an organism and which a foreign piece of DNA can be inserted for cloning purposes.
Examples:
Plasmids
Bacteriophage
Cosmids
Bacterial Artificial Chromosomes (BAC)
Yeast Artificial Chromosomes (YAC)

4. Plasmids
Plasmid: Small circular DNA that can replicate independently from a host’s chromosome.
Characteristics:
Smaller than chromosome (<1/20th the size)
Contain “non-essential genes”
Size varies 1 kb – 12,000 kb
Copy number is variable (1 – 200 copies per cell depending on the plasmid)
Some can integrate into the chromosome
Cloning vectors can carry up to ~15 kb
We’ll break down the anatomy of a plasmid cloning vector shortly!

5. Bacteriophage
Bacteriophage: A virus that infects and replicates within a bacterium.
Examples: λ phage, T2, T4, T7, T
Characteristics:
Head
Collar
Tail
Tail fibers
Base plate
50 kb
Approximately 10 – 25 kb can be replaced, up to 45 kb

6. Bacteriophage Vector

7. Select for antibiotic resistance
Cosmid Vector
Cosmids: Plasmid vectors that contain foreign DNA plus only cos sites
Characteristics:
- Hybrid between phage and plasmid
- Approximately kb can be replaced
Select for antibiotic resistance

8. BAC and YAC Vectors Bacterial Artificial Chromosome (BAC):
Based on the 7 kb F factor of E. coli
Can carry 100 – 300 kb
Yeast Artificial Chromosome (YAC):
Very high carrying capacity; up to 1000 kb
Resemble normal yeast chromosome: Telomeres, centromere
Yeast as a host (not E. coli as previous examples)

9. Cloning Vector Comparison
Cloning Capacity
Plasmids
15 kb
lambda Phage
10 – 25 kb
Cosmid
45 kb
BAC
100 – 300 kb
YAC
1000 kb

10. Plasmid vector -1 Features: 1. Origin of replication (Ori)
Recognition site for DNA Polymerase
Determines copy number (1 to >1,000)
“Relaxed” control via RNA
“Stringent” control via proteins
2. Selectivity marker
Resistance to antibiotic (ex. B-lactamases that break down ampicillin and penicillin
3. Multiple cloning site (MCS)
Unique restriction enzyme recognition sites
Downstream of promoter sequence
4. Promoter sequence
Upstream of the coding region
Landing site for RNA polymerase so that the gene can be transcribed into mRNA

11. Incompatibility Group
Plasmid Vector - 2
Features:
1. Origin of replication (Ori)
Recognition site for DNA Polymerase
Determines copy number (1 to >1,000)
“Relaxed” control via RNA
“Stringent” control via proteins
Common Vectors
Copy Number+
ORI
Incompatibility Group
Control
pUC ~
pMB1 (derivative) A
Relaxed
pBR322
~15-20
pMB1
pET
pGEX
pColE1
ColE1
pR6K
R6K B
Stringent
pACYC
~10
p15A
pSC101 ~5 C
pBluescript ~
ColE1 (derivative) and F1
pGEM
pUC and F1
2. Selectivity marker
Resistance to antibiotic (ex. B-lactamases that break down ampicillin and penicillin
3. Multiple cloning site (MCS)
Unique restriction enzyme recognition sites
Downstream of promoter sequence
Source:
How to choose?
Will plasmid be exclusively used in E. coli?
Wil you have more than one plasmid?
Do you need a high copy number?
Will your gene product be toxic?
Decision depends on copy number desired, host or hosts intended, compatibility.
Plasmids from same group shouldn’t be co-transformed because they compete for the same machinery
4. Promoter sequence
Upstream of the coding region
Landing site for RNA polymerase so that the gene can be transcribed into mRNA

12. Working concentration
Plasmid Vector - 3
Features:
1. Origin of replication (Ori)
Recognition site for DNA Polymerase
Determines copy number (1 to >1,000)
“Relaxed” control via RNA
“Stringent” control via proteins
Name
Class
Mode of Action
Working concentration
Kanamycin
Aminoglycoside
Binds 30S ribosomal subunit, causes mistranslation
μg/ml
Spectinomycin
Binds 30S ribosomal subunit, interrupts protein synthesis
μg/ml
Ampicillin
Beta-lactam
Inhibits cell wall synthesis
μg/ml
Erythromycin
Macrolide
Blocks 50S ribosomal subunit, inhibits aminoacyl translocation
μg/ml in Ethanol
Tetracycline
tetracyclin
Binds 30S ribosomal subunit, inhibits protein synthesis (elongation step)
10 μg/ml
Chloramphenicol
Binds 50S ribosomal subunit, inhibits peptidyl translocation
5-25 μg/ml in Ethanol
2. Selectivity marker
Resistance to antibiotic (ex. β-lactamases that inhibit cell wall; ampicillin and penicillin)
3. Multiple cloning site (MCS)
Unique restriction enzyme recognition sites
Downstream of promoter sequence
4. Promoter sequence
Upstream of the coding region
Landing site for RNA polymerase so that the gene can be transcribed into mRNA
Use fresh stocks! Antibiotics break down over time!
Store at -20 °C
Know which diluent to use; water or ethanol?
Do not add to hot media
There may be more than one!

13. Antibiotics Targets DNA Replication (Quinolones – Ciprofloxacin)
Metabolism (Sulfonamides - Trimethoprim)
Cell Wall (β-lactams – Penicillin
Glycopeptides - Vancomycin)
Protein Synthesis
(Tetracyclines,
Macrolides - Erythromycin
Aminoglycosides - Neomycin)

14. Plasmid Vector - 4 Features: 1. Origin of replication (Ori)
Recognition site for DNA Polymerase
Determines copy number (1 to >1,000)
“Relaxed” control via RNA
“Stringent” control via proteins
2. Selectivity marker
Resistance to antibiotic (ex. B-lactamases that break down ampicillin and penicillin
3. Multiple cloning site (MCS)
Unique restriction enzyme recognition sites
Downstream of promoter sequence
4. Promoter sequence
Upstream of the coding region
Landing site for RNA polymerase so that the gene can be transcribed into mRNA
Source: Novagen

15. Plasmid vector -5 pMB1 pBR322 (LacR Binding site)
Shine-Dalgarno sequence

16. Plasmid vector - 6 Features: 1. Origin of replication (Ori)
Recognition site for DNA Polymerase
Determines copy number (1 to >1,000)
“Relaxed” control via RNA
“Stringent” control via proteins
2. Selectivity marker
Resistance to antibiotic (ex. B-lactamases that break down ampicillin and penicillin
3. Multiple cloning site (MCS)
Unique restriction enzyme recognition sites
Downstream of promoter sequence
4. Promoter sequence
Upstream of the coding region
Landing site for RNA polymerase so that the gene can be transcribed into mRNA

17. Plasmids vector - 7 pMB1 pBR322 (LacR Binding site)
Shine-Dalgarno sequence
Shine-Dalgarno sequence

18. β-galactosidase β-galactosidase Oxidation Dimerization β-galactosidase
5-Bromo-4-Chloro-3-indoyl-β-D-galactopyranoside
(X-gal)
5-Bromo-6-Chloro-3-indoxyl
5,5'-dibromo-4,4'-dichloro-indigo
Absorbs at 615 nm
Blue Precipitate
β-galactosidase
β-D-galactose
o-Nitrophenol
Orthonitrophenalgalctopyranoside (ONPG)

19. βlue-White Colony Screen -1

20. βlue-White Colony Screen -2
X-gal +
IPTG plates

21. Catabolite Repression
β-galactosidase
In the presence of glucose, a cell does not need β-galactosidase
Secondary energy source
Primary energy source

22. Lac Operon lac Z – β-galactosidase lacY – Permease
lacA – Transacetylase
lacI – Lac Repressor
lacO – Operator – site on DNA to which the repressor binds
lacP – Promoter – site where RNA Polymerase binds

23. Isopropyl –D – 1 – thiogalactoside
Lac Operon Inducers
Isopropyl –D – 1 – thiogalactoside
(IPTG)
IPTG
E. Coli using this type of induction system has a deletion of the lacZ gene

24. Lac Repressor Binding site (Operator)
Plasmids vector
Lac Repressor Binding site (Operator)
Blue-White Screen
pMB1
pBR322
Shine-Dalgarno sequence

25. T7 Promoter Sequence from the T7 bacteriophage
T7 Polymerase is under control of the lac promoter
Expression is induced by the addition of IPTG
Transcription of T7 promoter is initiated by T7 Pol
T7 DNA Polymerase necessary for expression of insert gene in plasmid
How do we make T7 Pol to express our protein?
Use strains with λ(DE3) which means that these E. coli cells have prophage (Engineered phage) that has T7 RNA polymerase controlled by Lac regulatory construct. Ex) BL21 λDE3 Which you have already used in lab!

26. BL21 DE3 Host Cell

27. BL21 DE3 Host Cell

28. Polymerase Chain Reaction
polymerase chain reaction (PCR) is a method of amplifying a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
1. DNA is heated to 90°C to separate the two DNA strands
2. DNA is cooled to 30-65°C to allow primer annealing.
3. Reaction is heated to 72°C to allow DNA polymerase to effectively synthesize new DNA strands, creating two new dsDNA molecules.
4. The cycle is repeated, each time the amount of DNA doubles.
Forward Primer
Reverse Primer
1. Melting
2. Annealing
3. Elongation
4. Repeat cycle ~ 30 times

29. Primer Design -1 Three Hydrogen Bonds
Design primers that will dictate the region of interest
We need two primers in PCR:
Forward
Reverse
Two Hydrogen Bonds

30. Primer Design: G – C Content
Factors to consider:
Primers are typically around 18 – 22 bp long
Tm – Melting Temperature
Tm between 55 – 65 °C typically works best
Tm of both primers should be within 5 °C of each other, the closer the better!
Annealing temperature (Ta) is generally 5 °C lower than Tm
Primers that have less G – C bonds will have a lower Tm than a similar length primer with more G – C bonds.
G – C content target is ~45 – 55%
For sequences longer than 13 nucleotides:
     Tm= *(yG+zC-16.4)/(wA+xT+yG+zC)
Additional Factors to consider:
Primers price depends on size; 21 bp > 20 bp (but they’re cheap unless tags are included on the primer)
Primers larger than 60 bp become very expensive since synthesis becomes difficult and costs for validation increase
Three Hydrogen Bonds
(Stronger bond)
Two Hydrogen Bonds
(Weaker bond)

31. Primer Design - 2 3’ 5’ 3’ 5’ DNA Sequence
GCAATGCACTACGATTCGATCTCATGACATC
DNA Sequence
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Complementary DNA sequence
DNA is read in the 5’ to 3’ direction (unless specified)
DNA Polymerase travels in the 3’ to the 5 ‘ direction on the template strand
New DNA strand is synthesized from 5’ to 3’

32. Primer Design - 3 5’ 3’ 5’ 3’ 3’ 5’ DNA Sequence
GCAATGCACTACGATTCGATCTCATGACATC
DNA Sequence
Better annealing if 3’ end is a C or G (more H-bonds = more “sticky”!)
Helps anchor down DNA Polymerase when it comes in
At least one but avoid more than 3 “G – C clamps” at 3’ end 5’ 3’
GCAATGCACATACGATTCGAT
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Complementary DNA sequence

33. Primer Design - 4 3’ 5’ 5’ 3’ 3’ 5’ DNA Sequence
GCAATGCACTACGATTCGATCTCATGACATC
DNA Sequence
DNA Pol
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Complementary DNA sequence

34. Primer Design - 5 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ DNA Sequence
GCAATGCACTACGATTCGATCTCATGACATC
DNA Sequence
GCTAAGCTAGAGTACTGTAG
DNA Pol 3’ 5’
Reverse Primer
DNA Pol
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Complementary DNA sequence

35. Primer Design - 6 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ DNA Sequence
GCAATGCACTACGATTCGATCTCATGACATC
DNA Sequence
GCTAAGCTAGAGTACTGTAG
DNA Pol 3’ 5’
Reverse Primer
DNA Pol
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Complementary DNA sequence
Ordering Primers:
ALWAYS ORDER 5’ to 3’ DIRECTION!
Forward Primer: 5’ – GCAATGCACATACGATTCGATC – 3’
Reverse Primer: 5’ – GATGTCATGAGATCGAATCG – 3’

36. Primer Design: What to Avoid?
Secondary Structures:
Minimize intramolecular homology
“Hairpin loops”
Interfere with annealing temperature
Primer Dimers:
Primers should not contain sequences that allow one primer to anneal to another primer
ΔG of -7 kcal/mol or higher
Cross homology: - Primer homology to non-target sequences
Example: ΔG of -3.1 kcal/mol
Example: ΔG of -1.6 kcal/mol

37. Primer Design: Example 1
link

38. Primer Design: Example 2
Tm is well above hairpin loop formation
link

39. Adding Sequences to a Primer
Gene of interest
Q: What if our gene of interest doesn’t have the restriction sites that we desire?
A: Design PCR primers with restriction sites that we want.

40. Adding Sequences to a Primer 1
Gene of interest
Q: What if our gene of interest doesn’t have the restriction sites that we desire?
A: Design PCR primers with restriction sites that we want.

41. Adding Sequences to a Primer 25’ 3’
GCAATGCACTACGATTCGATCTCATGACATC
GCTAAGCTAGAGTACTGTAG 3’ 5’
Reverse Primer
Add BamHI site here
Add NdeI site here
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’

42. Restriction Sequences
Source: New England Biolabs

43. Adding Restriction Sites to a Primer
BamHI 5’ 3’
GCAATGCACTACGATTCGATCTCATGACATC
GCTAAGCTAGAGTACTGTAG 3’ 5’
Reverse Primer
NdeI
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’

44. Adding Restriction Sites to a Primer 3
BamHI 5’ 3’
GCAATGCACTACGATTCGATCTCATGACATC
GCTAAGCTAGAGTACTGTAG 3’ 5’
Reverse Primer
NdeI
Forward Primer 5’ 3’
GCAATGCACATACGATTCGATC
CGTTACGTGTATGCTAAGCTAGAGTACTGTAG 3’ 5’
Many restriction enzymes need additional nucleotides at the terminal ends of a DNA fragment to “sit” properly before cutting at their recognition site
The number of nucleotides necessary depends on the enzyme

45. Cleavage Close to DNA Fragment
May add on arbitrary sequence to the ends of your primers
Stay within optimal G-C content!