In a good design, what makes a good expression vector?
- Strong promoter: Greater expression, more production of mRNA, more protein
- Controllable Promoter - and inducible promoter
- done by adding an operator.
- Ex: Lac I in the lac operon, producing an repressor protein binding to the promoter to keep it off while IPTG changes the conformation of the repressor protein to turn it on
In a good design, what kind of plasmid copy number do we desire? What else is important about the plasmid? What about the host cell?
- Plasmid copy number
- Desire a high copy number
- Plasmid stability
- The plasmid is considered a foreign DNA to the host and it could possibly be attacked
- Host-cell physiology.
- Our host cell is BL21DE3
- Codon bias: preference to use a specific codon to code for a specific amino acid.
What does the Lac I gene give?What does it have an affinity for? How is it expressed?
Lac I gene gives a repressor protein
* The repressor protein has a high affinity for the operator
* The Lac I gene is constitutively expressed
What is the shape of the E.coli genome? What has been added to the genome in out experiment? Is there an operator? What king of promoter? What does the gene in the genome produce? What is it controlled by and done by?
- It is a circular genome
- The genome in the host cell as been engineered in such a way that it has an added T7 gene 1
- There is also an operator
- There is also a lac promoter
- T7 gene 1 produces T7 RNA polymerase
- It is a lysogen, a virus that does not cause destruction to the cell.
- E.coli has E.coli RNA polymerase naturally
- The gene is controlled by the lac promoter, an inducible promoter.
- Must be induced
- Done through IPTG
Why the the CR gene need a host? How is the promoter activated and what activates it?
- Does not have a gene to produce T7 polymerase
- Also has an inducible promoter
- Must be induced
- Done through IPTG
What two things it the PET system does IPTG induce? How does it work?
- Induces both the Lac promoter (e.coli) and the T7 promoter (pet29bcr)
- IPTG binds to the repressor protein, changing the confirmation of the repressor protein, creating a repressor IPTG complex. The protein can no longer bind to the operator.
What does the repressor IPTG complex result in and allow? What is produced? Is it produced before or after transcription?
- Since the protein cannot bind to the operator, the operator site is free for RNA polymerase to move forward and transcribe.
- T7 RNA polymerase is produced from the E.coli gene
- Occurs before the transcription of pet29bCR
What and where does T7 RNA polymerase bind to?
- Binds to the T7 promoter on the pet29bCR and transcription occur.
Why does a leaky promoter occur? Why is it common and preferable in bacteria?
- Dependent on how strong the binding between the promoter and the gene
- The repressor and operator binding is not 100% tight
- Is common and preferable in bacteria.
- Assists with having resources for the biochemical reaction, prior to induction.
What are the 3 experiments in Immunoblotting?
o SDS page
o Western Blotting
o Immuno activity assay
What is done after expression of the protein?
Purification
When does purification start?
- Starts when you lyse the cell
What are the 3 ways you can lyse a cell?
Physical pressure through french press
Ultrasonication
Enzymatic lysis
Describe the french press method.
- If there are a lot of cells, you can apply a french press method where physical pressure is applied and the cell breaks
Define ultrasonication.
Use sound waves to break the cells
Define ad state the 4 features of enzymatic lysis.
- Enzymatic lysis: Break down the cells using various enzymes and buffers
- A good buffer is one maintaining the solution at a specific pH well.
- For small samples
- Centrifuge
- Take supernatant
What does SDS PAGE stand for?
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
What is Polyacrylamide used?
- Polyacrylamide has smaller pores allowing molecules to migrate
Why is SDS used?
- Proteins, unlike DNA which is negatively charged naturally, have differing charges due to the functional group.
- Therefore, we need to use SDS to unwind the protein and make the charge of the protein negative so it will migrate through gel.
What type of gel is in SDS PAGE and what does it do?
- The gel is a denaturing and breaks the non covalent bonds to unwind the protein
What is electrophoresis and how do molecules migrate?
Migration of the ions through a matrix in an electric field
- Charged molecules migrate in an electric field with a velocity proportional to its overall density, size, and shape.
What 2 things are molecular separation based on?
Sieving effect: the amount of space in the gel
- Proportional to the percentage of acrylamide percentage. Similar to agarose gel, the greater the percentage the smaller the pores.
Electrophoretic Mobility: depends on the charge.
- The larger the protein, the longer it takes to migrate, and need to make sure there is a uniform charge so it moves based on the charge.
What are the charges in SDS? What does it impart? What is the proportion of SDS to protein?
Sodium is negative and sulfate is positive
The sds imparts a negative charge to the protein
1.4 g SDS/g of Protein
What is the solubility of dodecyl? What occurs even after treatment with it? What is the protein coated with and why?
Dodecyl is hydrophobic
- Unwraps the protein by disrupting the 3d structure (tertiary structure) of the protein
The protein we will use is globular and has a tertiary structure and therefore needs to be unwrapped
- Even after treatment of dodecyl, due to disulfide bonds from the amino acid cysteine there are still bonds remaining
- The entire protein is coated in sulfate from the dodecyl treatment.
