DNA cloning Requirements for DNA cloning: (1) A self-replicating segment of DNA
bacteriophage, plasmid, vector
(2) A procedure to introduce foreign DNA into a functioning cell
transfection, electroporation
(3) A procedure for selecting cells that have incorporated this DNA
antibiotic resistance
Site-directed Mutagenesis gene insertion
Use of nucleases to cleave DNA at specific recognition sites
Site-directed Mutagenesis gene insertion An artificial self-replicating DNA vector Contains a selectable marker (ampicilin resistance)
Contains a polycloning site for gene insertion
Site-directed Mutagenesis gene cloning procedure 1. Locate specific cleavage sites at the beginning and end of the gene of interest 2. Treat the vector with the same nucleases to create a compatible opening 3. Mix and anneal the vector with the gene 4. Covalently couple the fragments (DNA ligase) 5. Insert into cells to replicate (transformation)
Site-directed Mutagenesis gene cloning procedure
Site-directed Mutagenesis gene cloning procedure
1. Plasmids typically contain an antibiotic resistance gene 2. After transformation the bacteria is grown on a plate which contains that antibiotic 3. Only cells that have incorporated the plasmid will be capable of growing to produce colonies
Site-directed Mutagenesis PCR cloning Design two sets of primers, one set containing the desired mutation Extend each primer with DNA polymerase
Denature and re-anneal the DNA strands to produce heteroduplexes Only one heteroduplex can be extended from 3 to 5
Site-directed Mutagenesis Gateway cloning (1) insert the gene into an entry vector ccdB attP1 attB1 gene attB2
gene attP2
attL1 attR1 ccdB attR2
attL2 Entry Clone KanR
Donor Vector KanR
BP Clonase II
(2) transfer the gene to an expression vector
gene attL1 Entry Clone KanR attL2
ccdB attR1 attR2 Destination Vector AmpR
attP1
ccdB attP2 Donor Vector
gene
attB1
attB2 Expression Clone AmpR
LR Clonase II
KanR
Site-directed Mutagenesis experimental protocol 1. Pick target amino acid to be changed chemical modification pH studies sequence homology
2. Design a synthetic oligonucleotide to mutate the target amino acid
3. Use this primer to synthesize double stranded DNA 4. Verify production of the mutation 5. Characterize the new enzyme
Site-directed Mutagenesis verifying the mutation 1. Failure to grow if this is an essential enzyme for survival of the organism then creating a non-functional mutation should impair survival
2. Gene sequencing sequence the DNA to verify that the base changes have been introduced
3. Restriction mapping use the creation of a new restriction site or the elimination of an existing one to verify the mutation
Site-directed Mutagenesis restriction screening Allows selection of mutant enzymes through the creation or elimination of a restriction endonuclease site
The creation or elimination of a site changes the size of the DNA fragments obtained
Site-directed Mutagenesis characterization of mutant enzymes 1. Does the mutation affect any of the properties of the enzyme ? kinetics substrate binding stability
2. What is the magnitude of the effects ?
does a conservative replacement lead to large effects ? does a less conservative replacement increase the effect ?
3. What conclusions can be drawn about the role of the amino acid that has been mutated ?
Site-directed Mutagenesis kinetic characterization of mutant enzymes
Conclusions Cys135 provides an important catalytic functional group Gln162 does not appear to be absolutely essential Arg267 plays a role in substrate binding
Site-directed Mutagenesis Expanded approaches Three difference experimental approaches have been used to site specifically incorporate unnatural amino acids into enzymes:
1. Peptide ligation Synthesize a peptide containing an unnatural amino acid and then selectively ligate it between two fragments of the polypeptide
2. Chemical modulation Use mutagenesis to introduce a reactive amino acid and the selectively modify it with a series of amino acid analogue reagents
3. Supressor mutagenesis Introduce a stop codon at a specific site and then use a modified tRNA to incorporate unnatural amino acids
Site-directed Mutagenesis any amino acid in a protein can be selectively replaced with another amino acid the replacements are made at the genetic level by modifying the codon to incorporate the new amino acid characterizing the mutant enzyme that is obtained will provide information on the role of the amino acid that has been replaced the only unequivocal result from mutagenesis studies is when the mutation has no effect on the enzymes function
