Biotechnology lab 2 Flashcards
What is the objective of this lab?
Induce the expression of a recombinant protein in E. coli.
What is the first step to induce the expression of a recombinant protein?
The construction of a functional gene expression vector.
What is the most popular vector?
A plasmid.
What is a plasmid?
A circular DNA molecule that can replicated independently from the host chromosome.
How is a plasmid generally introduced?
Through transformation.
In this lab, how was a recombinant expression vector constructed?
By inserting the DNA sequence of the human PTEN into a pET vector.
What does the cloning vector pET contain?
Both the transcriptional and translational elements necessary to regulate the gene expression of PTEN.
What does the pET-PTEN expression vector contain?
A well characterized promoter located upstream of the multiple cloning site. It specifically contains: an ampicillin resistance marker, ColE1 origin of replication, F1 origin of replication, lacI gene, T7 transcription promoter, Lac operator region upstream of the promoter, Multiple cloning site where PTEN was inserted, 6x His tag.
Where was PTEN inserted in the expression vector?
In the multiple cloning site.
What is the purpose of the 6xHis tag?
To allow purification of the target protein utilizing affinity chromatography.
Where is the 6x His tag located?
At the N-terminus of the PTEN sequence.
Where are the transformed bacteria grown?
In liquid media containing ampicillin.
Which cells will be able to survive the ampicillin media?
Only cells containing the PTEN plasmid which expresses B-lactamase, allowing for ampicillin resistance will be able to propagate.
What do the two origins of replication allow?
For multiple copies of the vector to be produced.
What does the genetic engineering of the vector so it contains both the lac promoter and operator allow?
Allow the artificial induction of gene expression by the addition of a lactose analogue, IPTG, which binds to lacI (the lac repressor (transcriptional)) to allow RNA polymerase access to the promoter.
What is the T7 transcriptional promoter designed to be specific to?
The T7 RNA polymerase.
What is the disadvantage of using a bacterial host system?
The lack of a mammalian post-translational modification system that may be imperative to protein folding and activity.
In this lab, we will be exploiting the bacterial expression system in E. coli to produce what?
Large quantities of PTEN.
What is the bacterial cell line that will be used?
BL21 (DE3)-Codon plus-RIL competent cell line.
What is special about this cell line?
They contain additional copies of rare tRNAs that when lacking, can normally limit the translation of proteins in conventional E.coli strains.
What do the argU, ileY annd leuW tRNA genes encode?
tRNAs that recongize arginine, isoleucine and leucine codons enabling for improved heterologous protein production that may be otherwise restricted in A-T rich genomes.
What do the Ion and ompT proteases do?
Degrade proteins during recombinant purification.
Why do we need to use the Victor plate reader?
Use to measure the absorbance which is used to determine if the bacteria are in the log state of growth.
What is the point of the ColE1-compatible pACYC?
confers resistance to chloramphenicol which is required in the medium to maintain the pACYC plasmid
What does the endonuclease 1 (endA) do?
degrades DNA in miniprep procedures.
What reagent is used to clean the sonic micro tip?
Ethanol
What is done to separate the soluble proteins from the non-soluble ones?
centrifugation at 20000g in a pre chilled (4 degrees C) floor mounted centrifuge for 15 minutes.
