Expression Vectors and COmmon Techniques Flashcards
(36 cards)
What do expression vectors yield?
protein products of cloned genes
What two elements are required for active gene expression in a bacterial expression vector?
- strong promoter
2. ribosome binding site near an initiating AUG codon (ATG in DNA)
What is the rationale behind pairing expression vectors with very strong promoters?
the more mRNA that is produced the more protein product will be made and expression vectors main function id to yield the product of a gene
WHat are oligohistidine expression vector also called?
pTreHis
Why is it normally an advantage to keep a cloned gene repressed until it is time to express it? How do you keep a cloned gene turned off?
EUkaryotic protein produced in large quantities can be toxic even if the proteins are not actually toxic they can build up in large quantites and interfere with bacterial growth.
The protein may form insoluble aggregates called inclusion bodies.
Keeping the cloned gene downstream of an inducible promoter helps keep it turned off
Oligohistidine expression vectors have a short sequence just upstream of the multiple cloning sites that encode a stretch of what? and what will a protein be that is expressed in such a vector?
six histidines
And the protein will be a fusion protein with six histidines at its amino end.
What do oligohistidine regions like this have a high affinity for? and how can regions like these be purified?
They have a high affinity for divalent metal ions such as nickel (Ni2+) and they can be purified using nickel AFFINITY CHROMATOGRAPHY which is simple and fast.
What is a histidine analog called?
Imidazole
What is the orocedure that allows one to harvest essentailly pure fusion protein in one step and how is this possible?
Procedure: Bacteria make fusion proteins, lyse the fusion proteins, crude bacteria extract is added to a nickel affinity column, unbound proteins are washed out, and then the fusion proteins are washed out with histidine or a histidine analog
This is possible because few is any natural fusion proteins have oligohistidine regions so the fusion protein is essentially the only one that fins to the column.
What are some of the advantages of foreign genes produced in eukaryotic cells rather than prokaryotes
- Eukaryotic proteins tend to be folded properly so they are soluble rather than aggregated into inclusion bodies
- Eukaryotic proteins are modified in a eukaryotic manner
What is electrophoresis?
A technique that separates molecules by charge and/or mass in a gel matrix through which an electric current is passed
How is DNA separated by gel electrophoresis?
By charge: DNA is negatively charge at neutral pH and will migrate towards the positive pole
By size: little frictional drag on smaller DNAs and greater frictional drag on larger DNAs
How do you figure out the size of the fragments in gel electrophoresis and how do you see the individual fragments?
Run a size ladder and the protein inside the gel is stained.
WHat is pulsed field electrophoresis used to separate?
very large DNAs such a chromosomes
How is the current applied in pulse field electrophoresis?
The current is applied in pulses in the “forward” direction, interspersed with brief pulses of current in the reverse direction.
What is autoradiography?
It is a technique used to detect radio-labeled molecules by exposing a piece of X-ray film to a gel or other matrix that contains radio-labeled molecules
What is an example of how a vector can work as an expression vector thus resulting in a fusion protein?
The pUC and the pBS vectors place inserted DNA under the control of the lac promoter which lies upstream of the MCS. If an inserted DNA happens to be in the same reading frame as the lacZ’ interrupts then a fusion protein will result.
What does the lambda gt11 vector produce and how are they detected?
They produce fusion proteins that can be detected in plaques with a specific antiserum
What is fluorography?
A techniques similar to radiography but fluorography os used if the radioactive material is a low energy emitter
how does fluorography work?
FOr example tritium is a radioactive form of hydrogen that emits low energy praticles that can not barely make it out of the gel let alone an Xray film. The gel is soaked in a solution containing fluor, it will de-excite. This is an indirect way to locate the radioactivity and exposing the x ray film.
What are the resolution differences between autoradiography and fluorography?
Better resolution in fluorography.
What is technique that is in the category of phosphor imaging?
Scintillation Counting
What is Scintillation Counting?
Quantitative determination of radioactivity
How does Scintillation Counting work?
A cut band of the gel is placed into scintillation fluid that contains flour molecules which convert the radioactivity into visible light. Lowered into photomultipler tube that detects light resulting form the radioactive emmissions of the exciting fluor. Each burst of light or scintillation is recorded as counts per minute (cpm).
