Lab 2 - Recombinant Protein Expression in E. coli Flashcards
What are the tasks of this lab?
- Quantitation of log phase bacterial growth
- Protein expression induction
- Harvesting of bacterial cells by centrifugation
- Cellular lysis by sonication
- Bradford assay and preparation of samples for SDS-PAGE analysis
What is the objective of this lab?
To induce the expression of a recombinant protein in E. coli
What is important to understand prior to starting this lab?
How the expression of a cloned gene can produce a functional recombinant protein
What is the first step to expressing a cloned gene to produce a functional recombinant protein?
Construct a functional gene expression vector
What is the most popular cloning vector?
A plasmid
What is a plasmid?
A plasmid is a circular DNA molecule which has the ability to replicate without the interaction of a host chromosome
How is a genetically engineered plasmid introduced into a host system?
By a transformation process
For this lab, what has already been constructed for you and how?
A recombinant expression vector by inserting the DNA sequence of the human phosphatase and tensin homolog (PTEN) into a pET vector
What does the pET vector 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 (3’) of the multiple cloning site
What specific functional elements does the pET-PTEN expression vector contain?
- Ampicillin (Ap) resistance marker
- ColE1 origin of replication
- F1 origin of replication
- lacI gene
- T7 transcription promoter
- Lac operator up-stream of the promoter
- Multiple cloning site (where PTEN was inserted)
In addition to the functional elements, what else does the pET-PTEN expression vector contain and why?
His6 tag located at the N-terminus of the PTEN sequence to allow for purification of the target protein utilizing affinity chromatography
What are the transformed bacterial cells grown in?
Liquid media containing ampicillin
What cells allow for ampicillin resistance and propagate?
Only cells containing the PTEN plasmid which expresses beta-lactamase
What do the two origins of replication on the vector allow for?
Numerous copies of the vector to be produced
What does the genetic engineering of the vector to contain a lac promoter (Plac) and associate lac operator region allow for?
The artificial induction of gene expression by the addition of a lactose analog, isopropylthio-beta-D-galactoside (IPTG) which binds to a transcriptional repressor (LacI) to allow the RNA polymerase access to the promoter
What is the T7 transcriptional promoter designed to be specific for?
T7 RNA polymerase
What is the polymerase derived from?
The host bacterium which contains the gene for T7 RNA polymerase integrated into its chromosome
What can the lactose analogue IPTG induce?
The production of the target protein to 50% of cells total protein output within a few hours of induction by virtue of the Lac I allosteric mechanism
What will we be exploiting in this lab?
A bacterial expression system in E. coli to produce large quantities of our target protein, PTEN
What is the E. coli produced in milligram quantities of desired proteins used for?
- Enzyme assays
- Structure/function studies
- To produce antibodies
What is the disadvantage of using a bacterial host system?
The lack of a mammalian post-translational modification system that may be imperative for proper folding and activity of mammalian proteins
What bacterial cells line with be used in this laboratory?
BL21(DE3)-CodonPlus-RIL
What are the BL21(DE3)-CodonPlus-RIL cells specifically engineered to contain?
Additional copies of rare tRNAs that recognize the Arginine codons, the Isoleucine codon, and the Leucine codon, enabling for improved heterologous protein production that may otherwise be restricted or limited in AT-rich genomes