Final 3 Flashcards
What are the strategies for good over expression?
Good design
Plasmid copy number
Plasmid stability
Host cell physiology
What are the two types of promoters in the choice of an expression vector. Name and define the. Give an example for the second one.
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
What plasmid copy number do we desire?
Desire a high copy number
Why is plasmid stability important?
The plasmid is considered a foreign DNA to the host and it could possibly be attacked
In host cell physiology, what host are we using and what do we need to consider?
Our host cell is BL21DE3
Codon bias: preference to use a specific codon to code for a specific amino acid.
Study pet system diagram and be a ble to explain it
What is the pet system? Where are the components located? How are they introduced? What does the system look like before the introduction?
Pet system: The relationship between the BL21DE3 host cell and Pet29CR
All the above components are within the host cell.
The pet29CR was introduced to the host cell by transformation
Only the e.coli genome was present before transformation.
What does Lac I give? What does is have an affinity for? What type of expression is it?
The repressor protein has a high affinity for the operator
The Lac I gene is constitutively expressed
What shape is the e.coli genome? What is engineered in it? What are the two control elements? What does the engineered gene do? What RNA polymerase does E.coli have naturally? How is the gene controlled and how is it done?
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
What does pet29CR not have that requires us to use a host cell? What type of promoter does it have and how does it work?
Does not have a gene to produce T7 polymerase
Also has an inducible promoter
Must be induced
Done through IPTG
What does IPTG do in the pet system? How does IPTG work in the pet system?
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 happens to the operator when the repressor IPTG complex occurs? What is produced and from where? Does it occur before or after the transcription of pet29bCR?
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
Where does T7 RNA Polymerase bind?
Binds to the T7 promoter on the pet29bCR and transcription occur.
What is a leaky promoter dependent on? Why does it 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.
