Plasmids
engineered vectors
-circular, extra chromosomal DNA
What are they two different types of plasmid
Cloning vector-used primarily to propagate a DNA of interest
Expression vector-used primarily to express protein from a DNA of interest (gene)
What are common features of both types of plasmid?
-origin of replication- region of DNA that controls replication of the plasmid
-selectable marker- a gene which confers a growth advantage under selective conditions. It is important not to disrupt the selectable marker when cloning
(EX: the gene encoding beta-galactose metabolizes ampicillin and allows growth in the presence of this antibiotic)
-Multiple cloning site (polylinker)- region of DNA engineered to carry many useful RE sites. Designed to make it easier to find suitable RE sites for cloning your DNA of interest. Many RE sites in the polylinker cut only at that position
Screenable marker
a gene which allows distinguishing bacteria carrying a plasmid that contains an insert versus plasmid that does not contain a insert. A screenable marker allows you to identify bacteria carrying a plasmid with an insert (true positive), amid bacteria carrying the plasmid without an insert (false positives)
Examples of a screenable marker?
B-galactosidase= is an enzyme encoded by the gene lacZ
- its function in nature is to cleave lactose
- can also cleave the chemical X-gal to form a blue precipitate
- If B-galactosidase is made: X-gal is cleaved and blue precipitator forms
- If B-galactosidase is NOT made: X-gal remains uncleaved and NO precipitate forms
- THIS system works well in cloning vector not expression vectors because this strategy is not well suited for expression of the inserted gene
For plasmids what is an optional feature of both types of vectors?
Regulated gene expression- regions of DNA that control gene expression, limiting expression to specific conditions
- allows you to turn expression of a gene ON or OFF, often depending on the addition of a specific factor otthe growth media
- -lac operon: regulated gene rexpression
Expression vectors common components wit cloning vectors and ones that are necessary to transcribe and translate your gene of interest?
Basic features:
- origins of replication (often lower copy number than cloning vector)
- selectable marker
- multiple cloning site
- USUALLY NO SCREENABLE MARKER, because these are rarely compatible with expression of the inserted gene
Expression features:
- promoter- drives transcription of gene
- Ribosome binding site- present near start of transcript and signals ribosome bind and scan to start codon
- Start codon(ATG)- nucleotide triplet that signals start of translation
- Multiple cloning site (polylinker)- designed to allow insertion in a predictable reading frame and orientation.
- Fusion tag- can aid in purification, detection, etc. of protein- not always desired/present because it changes the protein!
What can expression vectors also do in organisms other than the originating organism?
synthesis of proteins:
- Pharmaceutical/industrial application
- Biochemical/functional analysis of protein
Properties of pET-41A using the T7 promoter to express the inserted gene in bacteria
- T7 promoter is from the T7 bacteriaphage
- T7 RNA polymerase is required for promoter activity
- E. Coli strains labeled “DE3” contain the T7 RNA polymerase
- pET-41a is designed to produce fusion protein
- The plasmid has the carboxy terminus of GST positioned before the gene
- We are fusing our protein with GST bc it will help us with purifying our protein later
- ORIENTATION AND READING FRAME of the insert are critical
- if the orientation of the gene is backward- the correct protein will not be produced
- if the gene is not inserted into the correct reading frame- the correct protein will NOT be produced
Inducible promoter (two conflicting desires make tight regulation necessary”
- High level of expression is generally desired- Purification of a large quantity of protein from a small quantity of cells is desired
- Healthy cells that grow well are desired. High levels of expression of recombinant protein can be lethal/detrimental to cell growth.
- Need a method of regulation or “switch” with which:
1. cells can be grown in the absence of (or with very limited) recombinant protein expression
2. Recombinant protein expression can be rapidly “turned on” after cells have grown
pET-41a and lac operon
what happens during normal growth conditions and induced growth conditions
- normal growth conditions, gene expression is repressed by production of lacI.
- Under induced growth conditions- gene expression is activated by removal of lacI inhibition
Repressed State:
steric hindrance with the bound lac repressor (lacI) prevents RNA polymerase from being able to transcribe the gene
Depressed State:
inducer moleclue binds LacI; LacI no longer can bind lac operate site and RNA polymerase can bind and transcribe gene
What are the common reasons for making fusion proteins (advantages)
one protein can be used to TAG another protein- allows VISUALIZATION or purification
- one protein can make another protein more SOLUBLE-allow biochemical analysis of an insoluble protein
- one protein can make another protein more STABLE-extend the function life of a protein
What are disadvantage of fusing two proteins?
- fusion may result in ALTERED folding- this can greatly affect function (often results in loss of function)
- Fusion changes the immunological properties of the protein- antibodies will recognize both parts of a fusion protein
Features of a gerne
- an open reading frame (ORF) begins with a start codon, has a span of successive ‘readable’ codons and ends with a stop codon
- The first ATG(AUG) within the ORF serves as a start codon, subsequent ATGs (AUGs) encode methionine)
- A stop codon is only functional if it is in frame with the start codon
- CODon- triplet of DNA bases that determine the amino acid in protein sequence
- Start codon: AUG (ATG)
- Stop codon: UGA(TGA), UAA(TAA), UAG(TAG)
What does a change in reading frame of a gene do?
alters translation of the remaining codons, resulting in incorrect amino acids and/or stop codons
Frame shifts
base insertions or deletions that result in the change of the reading frame
-CANNOT delete or insert a multiple of three
