Cloning Recombinant screening
Transformation of plasmids = very inefficient.
1. Construction of recombinant DNA Does host contain vector?
a. Cutting: restriction enzymes
b. Pasting: DNA ligase Ampicillin resistance gene in vector
2. Transformation ampicillin kills all bacteria that do not
a. Ca-buffer + heat shock contain vector
b. Electroporation
Does vector contain insert?
c. Bacteriophages (100% efficient)
d. RECOMBINANT SCREENING Selection systems
3. Proliferation o β-galactosidase gene
4. Isolation complementation
o suppressor tRNA gene
Restriction enzymes (endonucleases) Selection systems
e.g. HaeIII: Hemophilus aegypiticus III basic idea of all selection systems: insert interrupts
certain gene (RE cuts vector at multiple cloning
recognize usually 4-8bp palindrome sequence
site, the MCS, which is the recognition seq of the
notice difference between blund end and 5’ enzyme)
overhanging ends (sticky ends) below
β-galactosidase gene complementation (E. coli)
Plasmid with insert: white colonies
Plasmid without insert: blue colonies
Interrupted gene: LacZ
No β-galactosidase made
No blue product being formed out of X-gal
Some enzymes have same overhangs Suppressor tRNA
(compatibility) e.g. MboI & BamHI
Plasmid with insert: red colony
Length of the fragments cut depends on the Plasmid withour insert: white colony
recognition sequence Interrupted gene: suppressor tRNA
Special type of host that has nonsense
The longer the seq, the more rare, the
mutation -> mutant protein ->
longer the fragments
accumulation -> red colour
Cs and Gs are more rare than As and Ts
CG as a sequence (CpG islands) is very rare When gene is not interrupted, suppressor
because this gets methylated for tRNA is made, recognizing the stopcodon
epigenetic purposes inserting an AA, continuing translation -> WT
protein is made -> white colour
DNA ligase
Problem: recircularization!
Solved by:
Dephorylation of vector at 5’ end (ligation
only works fosfate-OH)
Use two different restriction enzymes
, Different vectors Lytic pathway
Classic plasmid (e.g. pUC19) – 0-10kB Cos-sites are 12bp 5’overhangs that seal in
Lambda phage host -> circular DNA
o Insertion – 0-10kB Early transcription facilitated by host RNA
o Replacement – 9-23kB polymerase
Cosmid/Fosmid – 30-45kB Coat proteins and replication machinery
P1 phage – 70-100kB made -> replication of DNA and formation
PAC – 130-150kB of virus particles
BAC – 150-300kB You get one long strain of viral DNA that is
YAC – up to 4 000kB cut at the cos-sites
M13 – max 3kB (ss) Cell lysis of host cell -> virus free
Phagemid – >10kB (ss)
Lysogenic pathway
Att gene has homologue in E.col
Recombination
Integration into host chromosome
= provirus
pUC19
In vitro packaging!
LacZ gene with MCS: β-galactosidase
recombinant screening = usage of a protein mix that contains coat
Ori = origin of replication -> can replicate proteins for packaging DNA with cos-sites into
independently of host genome, but needs phage particles
host replication machinery
AmpR: ampicillin resistance gene
Replacement vectors
You replace the non-essential part by an
insert
Note that Lambda phage NEEDS to contain
a chromosome of 37-50bp, not more/less!
o Advantage: when non-essential
part is cut, but no insert, no phage
particles are made
Lambda phage o Disadvantage: insert has under
= classic example of bacteriophage limit of about 10bp and upper
100% efficient transformation! limit of about 23bp
DNA is ds and linear in bacteriophage and Typically used for genomic DNA (gDNA)
gets circular in host
Insertion vectors
37-50kb genome
o Cos-sites! You leave the non-essential part as it is
o Protein coding part and just insert the insert
o Non-essential part with att gene* You have to use a vector that is relatively
Place for insert! short (37-40bp) -> 10kb space for insert
o DNA synsthesis and host lysis Typically used for coding DNA (cDNA)
regulation part
Lytic vs lysogenic pathway
Replacement vs insertion vectors
Transformation of plasmids = very inefficient.
1. Construction of recombinant DNA Does host contain vector?
a. Cutting: restriction enzymes
b. Pasting: DNA ligase Ampicillin resistance gene in vector
2. Transformation ampicillin kills all bacteria that do not
a. Ca-buffer + heat shock contain vector
b. Electroporation
Does vector contain insert?
c. Bacteriophages (100% efficient)
d. RECOMBINANT SCREENING Selection systems
3. Proliferation o β-galactosidase gene
4. Isolation complementation
o suppressor tRNA gene
Restriction enzymes (endonucleases) Selection systems
e.g. HaeIII: Hemophilus aegypiticus III basic idea of all selection systems: insert interrupts
certain gene (RE cuts vector at multiple cloning
recognize usually 4-8bp palindrome sequence
site, the MCS, which is the recognition seq of the
notice difference between blund end and 5’ enzyme)
overhanging ends (sticky ends) below
β-galactosidase gene complementation (E. coli)
Plasmid with insert: white colonies
Plasmid without insert: blue colonies
Interrupted gene: LacZ
No β-galactosidase made
No blue product being formed out of X-gal
Some enzymes have same overhangs Suppressor tRNA
(compatibility) e.g. MboI & BamHI
Plasmid with insert: red colony
Length of the fragments cut depends on the Plasmid withour insert: white colony
recognition sequence Interrupted gene: suppressor tRNA
Special type of host that has nonsense
The longer the seq, the more rare, the
mutation -> mutant protein ->
longer the fragments
accumulation -> red colour
Cs and Gs are more rare than As and Ts
CG as a sequence (CpG islands) is very rare When gene is not interrupted, suppressor
because this gets methylated for tRNA is made, recognizing the stopcodon
epigenetic purposes inserting an AA, continuing translation -> WT
protein is made -> white colour
DNA ligase
Problem: recircularization!
Solved by:
Dephorylation of vector at 5’ end (ligation
only works fosfate-OH)
Use two different restriction enzymes
, Different vectors Lytic pathway
Classic plasmid (e.g. pUC19) – 0-10kB Cos-sites are 12bp 5’overhangs that seal in
Lambda phage host -> circular DNA
o Insertion – 0-10kB Early transcription facilitated by host RNA
o Replacement – 9-23kB polymerase
Cosmid/Fosmid – 30-45kB Coat proteins and replication machinery
P1 phage – 70-100kB made -> replication of DNA and formation
PAC – 130-150kB of virus particles
BAC – 150-300kB You get one long strain of viral DNA that is
YAC – up to 4 000kB cut at the cos-sites
M13 – max 3kB (ss) Cell lysis of host cell -> virus free
Phagemid – >10kB (ss)
Lysogenic pathway
Att gene has homologue in E.col
Recombination
Integration into host chromosome
= provirus
pUC19
In vitro packaging!
LacZ gene with MCS: β-galactosidase
recombinant screening = usage of a protein mix that contains coat
Ori = origin of replication -> can replicate proteins for packaging DNA with cos-sites into
independently of host genome, but needs phage particles
host replication machinery
AmpR: ampicillin resistance gene
Replacement vectors
You replace the non-essential part by an
insert
Note that Lambda phage NEEDS to contain
a chromosome of 37-50bp, not more/less!
o Advantage: when non-essential
part is cut, but no insert, no phage
particles are made
Lambda phage o Disadvantage: insert has under
= classic example of bacteriophage limit of about 10bp and upper
100% efficient transformation! limit of about 23bp
DNA is ds and linear in bacteriophage and Typically used for genomic DNA (gDNA)
gets circular in host
Insertion vectors
37-50kb genome
o Cos-sites! You leave the non-essential part as it is
o Protein coding part and just insert the insert
o Non-essential part with att gene* You have to use a vector that is relatively
Place for insert! short (37-40bp) -> 10kb space for insert
o DNA synsthesis and host lysis Typically used for coding DNA (cDNA)
regulation part
Lytic vs lysogenic pathway
Replacement vs insertion vectors
