Genetic transformation of E.Coli and selection, DNA recombination without ligase: topoisomerase, cre-lox recombination, Gate way method
etc. DNA library: genomic library, cDNA library, expression library, substraction library
Mitesh Shrestha
Historical Perspective
• Frederick Griffith 1928 London – First controlled demonstration of genetic transformation
– Griffith made the observation that nonpathogenic bacteria (Streptococcus pneumoniae) became pathogenic when mixed with a virulent strain of heat-killed S. pneumoniae (i.e. injected mixture killed mice)
– The mechanism of transforming
nonpathogenic bacteria to deadly
bacteria was not known
• In 1944 Oswald Avery demonstrated
that DNA is responsible for conferring
pathogenic properties
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Antibiotic-sensitive bacterial cell
Plasmid DNA
Calcium chloride treatment permeablises cell wall
Transformed bacterial cell
Transformed bacteria selected by growing on solid
media containing appropriate antibiotic
Bacterial transformation
Competent Cells
• Competence is the ability of cells to take up exogenous DNA from the environment
• Two types of competence: – Natural competence: Bacteria have cellular machinery to take up DNA
from environment
– Artificial competence: Cells are made competent in the laboratory allowing them to take up DNA
Utilization of Competent Bacteria
• Heat Shock: – Drives DNA into cells
– Hold cells on ice in presence of CaCl2 to promote permeability of cells to plasmid DNA
– Cells are heat shocked at 42 ºC for 50 – 60 seconds to allow circular plasmid DNA to enter cells
• Electroporation: – Subject cells to electric shock to perforate membrane
– Plasmid DNA enters cells through temporary holes
– Efficient transformation of large plasmids
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E. coli advantages • Several different types of plasmid can be used
to introduce foreign DNA.
• Foreign DNA can account for up to 60% of its total protein production.
• Fast growing.
• Easy to transform.
• Easy to manipulate.
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E. coli disadvantages
• Does not carry out post-translational
modifications to proteins.
• These modifications are necessary for protein
function in, for example, human cells.
• Bacterium occurs naturally in the intestines of
humans and under certain circumstances can
cause disease.
DNA recombination without ligase
• Topoisomerase,
• Cre-lox recombination,
• Gate way method
Topoisomerase
• Budding yeast cells produce two type I topoisomerases, Top1 and Top3, and one type II topoisomerase, Top2. Mutation of TOP1 or TOP2 results in an increased rate of homologous recombination, but only at the ribosomal (r)DNA locus.
• Aside from this, top1 mutants display a surprisingly mild phenotype. In contrast, TOP2 is an essential gene, and top2 mutants cannot complete mitosis, because of a defect in chromosome segregation .
• Top1 and Top2 efficiently relax either negatively or positively supercoiled DNA, and the increased recombination seen in top1 and top2 mutants may be due to an accumulation of DNA torsional stress in the heavily transcribed rDNA. Top3, however, can relax only negatively supercoiled DNA, and then only weakly . Nevertheless, top3 mutants display a reduced growth rate and hyper-recombination that affects repeat sequences throughout the genome.
• In E. coli, there are two type I topoisomerases, topo I and topo III, encoded by the topA and topB genes, respectively. Both are structurally related to yeast Top3, and mutations in bacterial topB also result in hyper-recombination.
Cre-lox recombination
• The Cre-lox system is a technology that can be used to induce site-specific recombination events.
• The system consists of two components derived from the P1 bacteriophage: the Cre recombinase and a loxP recognition site.
• The P1 bacteriophage uses these components as part of its natural viral lifecycle, and researchers have adapted the components for use in genome manipulation.
Cre-lox recombination
• Cre recombinase, originally named because it “causes recombination” (although later referred to as the “cyclization recombinase”), is a 38 kDa protein responsible for intra- and inter-molecular recombination at the loxP recognition sites.
• A key advantage of the system is that Cre acts independently of any other accessory proteins or co-factors, thus allowing for broad applications in a variety of experiments.
Cre-lox recombination
• LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting of two 13-bp long palindromic repeats separated by an 8-bp long asymmetric core spacer sequence.
• The asymmetry in the core sequence gives the loxP site directionality, and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT.
• The loxP sequence does not occur naturally in any known genome other than P1 phage, and is long enough that there is virtually no chance of it occurring randomly. Therefore, inserting loxP sites at deliberate locations in a DNA sequence allows for very specific manipulations.
Cre-lox recombination
• The Cre recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either on the same or on separate pieces of DNA. Both 13bp repeat sequences on a single loxP site are recognized and bound by a Cre protein, forming a dimer.
• The two loxP sites then align in a parallel orientation, allowing the four Cre proteins to form a tetramer. A double-strand DNA break occurs within the core spacer of each loxP site and the two strands are ligated, resulting in the reciprocal crossover event.
Cre-lox recombination Outcomes
• Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination results in an inversion and the region of DNA between the loxP sites is reversed.
• Deletion: If the sites face in the same direction, the sequence between the loxP sites is excised as a circular piece of DNA (and is not maintained).
• Translocation: If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites.
Cre-lox recombination Outcomes
Cre-lox recombination
• Cre-dependent gene expression – placing a stop codon with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression in the absence of Cre. In the presence of Cre, the stop codon is excised, and gene expression proceeds.
• Cre-dependent gene knockout – conversely, putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP”), will permit gene expression until Cre is present, at which time the gene will be disrupted or deleted.
Cre-lox recombination
• Selection marker removal – in conventional mouse targeting, targeted clones are selected for using a resistance marker; however, it is often desirable to remove the marker after the initial selection process. By floxing the selection marker, Cre can be used to easily perform this eviction.
• Regulated Cre expression – placing Cre downstream of promoters that are active only in certain cell or tissue types, during certain stages of development, or by making the Cre inducible (such as with tamoxifen or doxycycline), the Cre recombinase can be expressed only in specified cells or at specified times. Combining this with some of the loxP methods described above, a genetic modification can be restricted based on experimental constraints. This has been used for a wide range of purposes, including activating an oncogene only in a particular organ, or bypassing embryonic lethality.
Gate way method
• Gateway BP reaction: PCR-product with flanking att B sites (this step can also use other methods of DNA isolation, such as restriction-digestion) + Donor vector containing att P sites + BP clonase => Gateway Entry clone, containing att L sites, flanking gene of interest
• Gateway LR reaction: Entry clone containing att L sites + Destination vector containing att R sites, and promoters and tags + LR clonase => Expression clone containing att B sites, flanking gene of interest, ready for gene expression.
Gate way method
Genomic library
construction
Screening a genomic library using DNA hybridization to a
(radio-)labeled DNA probe
Note: a cDNA is commonly (radio-)labeled and used as
a DNA probe to screen a genomic library
How many genomic clones must be screened to find your gene?
Theoretically, you will need to screen N clones where N=ln(1-P)/ln(1-f) where P=the probability of finding your gene and f=the average size of the cloned genomic sequence in your vector divided by the total genome size.
How many clones must you screen to find your gene in a human gene library packaged in EMBL 3 with 99% certainty?
N=ln(1-0.99)/ln(1-20kb/2.8 x 106kb)= 6.4 x 105 clones
2-8
Constructing a genomic library in phage
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Constructing a genomic library in cosmids
2-10
Constructing a genomic library in YACs
Construction of a cDNA library
2-2
Differences between a genomic and cDNA library
(Not in Chapter)
Genomic Library Promoters Introns Intergenic Non-expressed genes
cDNA Library Expressed genes Transcription start sites Open reading frames (ORFs) Splice points
Expression library
• Expression cloning is a technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein.
• Expression vectors are a specialized type of cloning vector in which the transcriptional and translational signals needed for the regulation of the gene of interest are included in the cloning vector.
• The transcriptional and translational signals may be synthetically created to make the expression of the gene of interest easier to regulate.
Immunological screening of an expression cDNA library with a primary antibody and labeled secondary antibody; note the label is often an enzyme label like alkaline phosphatase or
horseradish peroxidase, but it can also be 125I
Subtraction library
• For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful.
• A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type.
• This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable.
Method I
• mRNA from the target material is hybridized with first strand cDNA from the subtractor material immobilised on Dynabeads®.
• The subtracted mRNA is left in the supernatant after magnetic separation of beadbound subtractor cDNA with captured common mRNA, and the subtractor beads can be reused.
• After the final hybridisation step, the subtracted specific mRNA is reverse transcribed to radiolabelled cDNA and used to screen cDNA libraries or for cDNA cloning.
• When the difference between the subtractor and the target mRNA population is small, large amounts of target specific mRNA may be difficult to obtain and the PCRbased method of Lambert would be more appropriate.
Method II
• An alternative approach is to create immobilised cDNA libraries from both target and subtractor mRNA.
• Second strand cDNA is synthesised by random priming of target cDNA and the fragments eluted and mixed with excess immobilised subtractor cDNA.
• Common fragments are annealed and removed, while the unique fragments left in the supernatant are used as a probe to screen cDNA libraries.
• If the amount of mRNA is limited or the two mRNA sources are very similar, the material might be insufficient for several rounds of subtraction to be performed, or the material remaining for screening purposes might be insufficient.
• This can be solved by allowing the subtracted cDNA fragments to reanneal back onto the immobilised subtractor cDNA. The double stranded cDNA produced is then cut, linkers ligated and the fragments amplified by PCR
