Lecture 24 Flashcards
What is a vector?
DNA molecule that assists in gene transfer
What is a replicon?
Nucleic acid molecule capable of autonomous replication
What is a chromosome?
A DNA replicon that is essential for the growth of the cell under normal conditions
What is a plasmid?
An autonomously replicating extra chromosomal DNA molecule
What is the structure of the commonly used pMB1/ColE1 plasmid?
All have selectable markers
Have a multiple cloning site which is contained in a lac-alpha region
SP6/T3/T7 are bacteriophage promoters adjacent to multiple cloning site which allows transcription to occur
What are shuttle vectors?
Vectors which enable the transfer of genes between hosts
What are the important DNA regions to have on shuttle vector?
Selectable marker for each different type of host (i.e. one for E. Coli and one for yeast or mammalian cells)
An origin of replication, one for each different type of host
Lac alpha gene with multiple cloning site
What is important in any good plasma vector?
Must have a high copy number to ensure good levels of gene expression (in a good system it will be 25-50% of all cellular protein)
The expression of the gene must be inducible (to prevent overloading of the cell)
Fusion protein must be used for higher stability, solubility and easy purification (proteins may require an inclusion body if highly insoluble)
Post translational modifications may pose issues as these can be different between species
What are common methods of making a protein easy to purify from a plasmid expression vector?
A 6 histidine repeat allows the protein to bind to a nickel column
Or binding to other columns such as a glutathione column
A protease cleavage site is included to allow recovery of the protein
What are common methods of making the gene in a plasmid inducible?
Temperature sensitive lac where the repressor Ptac binds at 30 but not 37 degrees
Temperature sensitive T7 polymerase gene
Lac promoter induced by IPTG
What is Ptac?
A hybrid trp and lac promoter, high level of trp and repressible by lacI
What is used to clone large genomic fragments?
Several artificial chromosomes have been tried Bacterial Chromosomes (BACs) are typically the ones used for construction of genomic libraries
What are the features of BACs?
Good size of insert
High stability
Use of E. Coli rather than yeast
technically difficult to mae
How were BACs designed?
Plasmids with replication origin from the F factor of E. Coli
Low copy number (1-4/cell)
Stable inheritance due to genes for replication and partition
100-150kb inserts
