Cloning in E. coli Flashcards
What type of sequences do type II restriction enzymes recognise in DNA?
Symmetrical sequences (palindromes)
There are many type II restriction enzymes with different sequence specificities. How many enzymes and specificities are there?
> 2500 enzymes
>200 different specificities
Why are type II restriction enzymes known as endonucleases?
Because the cut dsDNA within the recognition sequence
What happens to the cut DNA when a type !! restriction enzymes cuts at an asymmetric site?
It leaves single stranded complementary ends (cohesive/sticky ends)
What sequence does the type II restriction enzyme EcoRI recognise (5’ to 3’)? Where does it cut within the sequence?
GAATTC
Cuts after the G base
What sequence does the type II restriction enzyme BamHI recognise (5’ to 3’)? Where does it cut within the sequence?
GGATCC
Cuts after the first G base
What sequence does the type II restriction enzyme EcoRV recognise (5’ to 3’)? Where does it cut within the sequence?
GATATG
Cuts after the first T base
What sequence does the type II restriction enzyme PstI recognise (5’ to 3’)? Where does it cut within the sequence?
CTGCAG
Cuts after the A base
What sequence does the type II restriction enzyme NotI recognise (5’ to 3’)? Where does it cut within the sequence?
GCGGCC
Cuts after the first C base
What sequence does the type II restriction enzymes Sau3A and MboI recognise (5’ to 3’)? Where does they cut within the sequence?
GATC
Cuts before the first G base
What are type II isoschizomer restriction enzymes?
Enzymes that recognise the same sequence
They can cut in the same way or sometimes cut differently
Give an example of type II isoschizomer restriction enzymes that cut in the same way
Sau3A and MboI
Give an example of type II isoschizomer restriction enzymes that cut differently
SmaI and XmaI
What sequence does the type II restriction enzyme SmaI recognise (5’ to 3’)? Where does it cut within the sequence?
CCCGGG
Cuts after the last C base
What sequence does the type II restriction enzyme XmaI recognise (5’ to 3’)? Where does it cut within the sequence?
CCCGGG
Cuts after the first C base
Give 3 examples of host effects in E. coli that cause DNA modification
Dam methylation
Dcm methylation
Mcr system
What sequence is recognised by Dam methylation (5’ to 3’)? What base in this sequence is methylated? And what enzymes can this modification block?
GATC
Methylates the A base
Blocks MboI and XbaI
What sequence is recognised by Dcm methylation (5’ to 3’)? What base in this sequence is methylated? And what enzymes can this modification block?
CCA/TGG
Methylates the last C base
Blocks EcoRII
What sequence is recognised by the Mcr system (5’ to 3’)? At what bases in this sequence does this modification cut methylated DNA at?
A/GCN(40-80)A/GC
Cuts at both C bases
What bonds do type II restriction enzymes cut and what does this leave at the ends of the fragment?
DNA phosphodiester bonds are cut to leave a 5’-phosphate and a 3’OH at the ends
(Fragments produced have the same ends)
Is ATP needed for type II restriction enzymes to break the DNA phosphodiester bonds?
No
What can happen to the ends of fragments cut by type II restriction enzymes if there are protruding overhangs?
The ends can reanneal
What is usually the frequency of restriction site cutting (in 50% GC DNA)?
1 restriction site per 4^N base pairs where N is the length of the recognition sequence
Why can’t you often use a complete digestion to clone a complete gene?
Because it would either:
Produce a fragment that is too large
Cut within the gene (once or many times)
How would you clone a complete gene?
Using a partial digestion and then clone ‘random’ fragments from the partial digestion
Describe the mechanism by which annealing of ‘sticky’ ends occurs
Ligate together by covalent ligation…
DNA ligase reseals DNA joining (5’-P to 3’-OH)
Requires ATP
Requires enzyme from phage T4
What is special about the reformed site of a recombinant DNA molecule?
It is re-cleavable by the same enzyme
What does the chance of a digested vector finding the end of another molecule or finding the other end of itself depend on?
Ligation conditions
Give three important factors when considering ligation conditions
The ratio of vector to insert DNAs
The size (length) of DNAs
The concentration of DNA
What happens when there is a low concentration of DNA in the ligation reaction?
Intramolecular ligation
I.e. the formation of vector circles
What happens when there is a high concentration of DNA in the ligation reaction?
Intermolecular ligation
I.e. the formation of recombinants
What happens when there is a very high concentration of DNA in the ligation reaction?
Multiple intermolecular ligations
I.e. the formation of multimers
What reagent can prevent vector religation?
Alkaline phosphatase
What does alkaline phosphatase do?
Removes the 5’-P to 5’-OH bonds
Give five essential features that need to be considered when choosing a cloning vector
Maintenance (replicon) Restriction enzyme site Its ntroduction into host Genetic selection marker A way to identify recombinants
Why do we need to consider maintenance (replicon) when choosing a cloning vector?
There needs to be free replication in the host of the plasmid or bacteriophage DNA
Copy number may need to be considered in the case of a plasmid vector
Why do we need to consider restriction enzyme sites when choosing a cloning vector?
For insertion of foreign DNA fragment
They need to be unique
Vector needs to have a multiple cloning site
Why do we need to consider introduction into host when choosing a cloning vector?
Need to choose whether to carry out a transformation (plasmids) or infection (phages)
Why do we need to consider genetic selection markers when choosing a cloning vector?
To recover cells with the vector or clones
To pick cells when there is a low percentage of transformants
Can use antibiotic resistance
Why do we need to consider the identification of recombinants when choosing a cloning vector?
As an easy (visual) screen for recombinants vs. the vector
Can be used for insertional inactivation screens
Give 3 features of the E. coli plasmid vector
Give 2 features of the E. coli PAC vector
P1 phage delivery
100-300 kb insert
Give 2 features of the E. coli lambda phage vector
5-25 kb insert
Efficient delivery by in vitro
Give 3 features of the E. coli BAC vector
Bacterial artificial chromosome
F plasmid origin
<300kb insert
Give 3 features of the E. coli cosmid vector
Plasmid-like with phage delivery
35-45 kb insert
In vitro packaging delivery
Give 3 features of the yeast YAC vector
Yeast artificial chromosome (linear DNA)
Yeast origin of replication
200-2000 kb insert
What does the pBR322 plasmid vector have from the ColE1-like plasmid pMB1, and what does this allow?
ori and rop
Copy number control
What is the copy number per cell of pBR322?
~15
What does the pBR322 plasmid vector have from the pSC101 plasmid?
Tetracycline resistance gene (Tc^R)
What does the pBR322 plasmid vector have from the Tn3?
Ampicillin resistance gene (Ap^R)
Does the pBR322 plasmid contain any unique restriction enzyme sites?
Yes
How can insertional inactivation be achieved in the pBR322 plasmid?
Tc^R/S using BamHI
Amp^R/S using PstI
What does the pUC19 plasmid vector have from the pMB1 plasmid?
ori
What is special about the copy control of the pUC19 plasmid?
It is mutated
delta rop
What is the copy number per cell of pUC19 at 37 degrees centigrade?
~75
What is the copy number per cell of pUC19 at 42 degrees centigrade?
~200
What does the pUC19 plasmid vector have from the Tn3?
Ampicillin resistance gene (Ap^R)
What does the pUC19 plasmid vector have from the E. coli chromosome?
lacZalpha
What is present in lacZalpha of pUC19?
Synthetic multiple cloning site (MCS)
How can you achieve insertional inactivation of lacZalpha in the pUC19 plasmid?
Using the MCS
Does the pUC19 plasmid contain any unique restriction enzyme sites?
Yes
What is a common way to identify recombinants?
Using blue-white screening
In the blue-white screen, what do white cells indicate?
No lacZ activity
In the blue-white screen, what do blue cells indicate?
lacZ activity
What does lacZ make?
Beta-galactosidase (beta-gal)
What does beta-gal do?
Cleaves colourless X-gal to blue indigo
What is the full name of X-gal?
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
What happens as a result of the insertion of DNA fragments into the MCS in the lacZalpha sequence of a plasmid?
Insertional inactivation
Beta-gal is no longer produced and so X-gal cannot be converted
What is the size of pUC vectors?
Only 2.7kb long
What do pUC plasmids contain?
ori (~0.6kb)
Amp^R (~1kb)
lacZalpha
How long is the native E. coli lacZ gene?
> 3kb long
How would you construct a genomic library?
Isolate chromosomal DNA
Digest chromosomal and vector DNA with restriction enzymes
Ligate chromosomal DNA with vector DNA
Transform ligated DNA into E. coli
Select cells containing vector, e.g. antibiotic medium
Screen for recombinants in library
All ligated DNA is maintained in E. coli, true or false?
False
When each transformant containing a vector or recombinant forms a colony of identical cells what are these cells known as?
Clones
When all cells of clones contain the same DNA molecule what are these known as?
Molecular clones
What is a clone collection better known as?
A library
A complete set is of the whole genome
Are clones kept together or separately?
Separately (sometimes pooled)
How would you ensure that a library is predominantly recombinants?
A simple screen to differentiate recombinants and vector alone, e.g. blue-white X-gal screen for pUC vectors
Can recombinants be identified using antibiotic medium if the vector alone is able to grown on antibiotic medium?
No, you would have to screen for the recombinants to identify the lack of inserts
Can recombinants be identified using antibiotic medium if the recombinant is able to grown on antibiotic medium?
Yes, the recombinant is identified by the screen
What two strategies could you use to identify a cloned fragment of DNA or a gene?
Physical (to detect the DNA itself)
Biological (to detect the expression of gene)
How could you physically identify a cloned fragment of DNA or a gene?
Screen using base sequence complementarity by hybridisation to a DNA probe or PCR
How could you biologically identify a cloned fragment of DNA or a gene?
Screen for expression phenotype by complementation of a mutation
Screen for protein product directly using antibodies or a specific biological assay
In what ways can you introduce DNA into an E. coli host cell?
Through chemical transformation or electroporation
Describe how electroporation works
Cells with DNA are in dH2O (non-conducting)
DNA enters cells via open pores in the membrane after a short, high amplitude electrical discharge is delivered
Describe how chemical transformation is carried out
Divalent metal ions permeabilise wall to DNA to create competent cells
DNA is added at 0 degrees centigrade and enters the cell after a brief heat shock
Allow time for gene expression and plate on selective medium to recover transformants
How many DNA molecules per cell gives a transformant clone when carrying out chemical transformation?
1 DNA molecule
When carrying out chemical transformation, how many transformants are produced per microgram of input DNA?
E6-E9 transformants
What percentage of cells are transformed when carrying out chemical transformation?
Very low percentage of cells are transformed and thus selection is needed
Give three ways in which plasmid DNA can be recovered from the E. coli host cell
Production of a cleared lysate
Affinity purification
Dye-bouyant equilibrium-density centrifugation
What is the basis of producing a cleared lysate to recover plasmid DNA form the E. coli host cell?
Small plasmid molecules are resistant to denaturation
What is the general procedure for production of a cleared lysate to recover plasmid DNA from the host E. coli cell?
Denature cell contents and fragment chromosome
Aggregate denatured materials by salt-precipitation
Spin to remove aggregate
Plasmids recovered in supernatant (cytosol)
How does affinity purification work to recover plasmid DNA from the host E. coli cell?
DNA, an anionic polymer, binds to:
Anion exchange resins, e.g. “Qiagen”
Silica or glass particles, e.g. “Geneclean/Glassmilk”
How does dye-bouyant equilibrium-density centrifugation work to recover plasmid DNA from the host E. coli cell?
EthBr intercalates into DNA, reduces density
More EthBr enters linear fragments
Plasmid DNA remains denser than chromosomal DNA
Plasmid DNA equilibrates and bands lower in gradient
What is the basis of dye-bouyant equilibrium-density centrifugation to recover plasmid DNA form the E. coli host cell?
CsCl-density gradient containing ethidium bromide (EthBr)
List 5 important genotypes of E. coli that need to be considered when using it as a host strain
Mcr endonuclease EndA (non-specific endonuclease) Dam and Dcm systems Rec (homologous recombination) Lon (long form of E. coli; encodes La protease)
What is the role of the Mcr endonuclease and what can we use it for?
Mcr cuts DNA at methylated-C in CpG and GpC motifs common in eukaryotic DNA
Use mcr mutant to maximise cloning efficiency
What is the role of EndA mutation?
endA mutation improves the quality of plasmid DNA preps
What can Dam and Dcm systems affect?
DNA-adenine methylase and DNA-cytosine methylase affects cutting by some enzymes
What is the role of different Rec?
recA stabilises plasmids carrying repeated sequences
recB/C abolishes exonuclease-V activity; stabilises IRs
sbcB abolishes exonuclease-1 activity
sbcB/C and recB/C strain is Rec+ but propagates IRs stably
recF(J) abolishes plasmid-plasmid homologous recombination
What is the role of Lon?
Prevents degradation of some eukaryotic proteins
Summarise the steps of gene cloning
- DNA is fragmented using restriction enzymes
- Genome library is created using PCR
- DNA is cut using restriction enzymes
- DNA and vector are joined using DNA ligase
- Selection using antibiotic resistance
- Introduction (transformation) of recombinant into host cell
- Recovery (isolation) of transformants
- Recombinant identification
