gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pET Bacterial Recombinant Protein Vector
The pET vector system is a powerful and widely used system for expressing recombinant proteins in E. coli. The gene of interest is cloned into the pET vector under the control of the strong bacteriophage T7 transcription and translation regulatory system. Activation of expression is achieved by providing T7 RNA polymerase within the cell. When the system is fully induced, nearly all of the cell’s resources are devoted to expressing the gene of interest. With just a few hours of induction, the recombinant protein could comprise nearly half of the cell’s total protein.
For further information about this vector system, please refer to the papers below.
|Methods Enzymol. 185:60-89 (1990)||Use of T7 RNA polymerase to direct expression of cloned genes.|
|J Mol Biol. 219:45 (1991)||Development of the T7lac promoter system.|
The gene of interest is initially cloned into the pET vector in a bacteria host that lacks the T7 RNA polymerase gene. This eliminates plasmid instability due to expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be initiated in two possible ways. The host cells can be infected with phage carrying the T7 RNA polymerase gene (e.g. λCE6 phage), or more commonly, the pET plasmid can be transferred into a bacteria host strain whose genome has been engineered to carry the T7 RNA polymerase gene under the control of theLacUV5 promoter. Expression of the T7 polymerase is induced by the addition of the lactose analog IPTG to the bacterial culture.
The pET vector exists as a low copy number plasmid in host E. coli, which reduces leaky expression before induction. The vector utilizes the T7lac promoter system for strong and tightly controlled gene expression. In this system, there is a T7 promoter that can be acted upon by T7 RNA polymerase to drive high-level expression of the gene of interest. Additionally, there is a lac operator (LacO) sequence just downstream of the T7 promoter that can be acted upon by the lac repressor (LacI) protein to block transcription of the T7 promoter. The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts at the LacUV5 promoter in the host cells to repress expression of the T7 RNA polymerase gene by the host polymerase, and also functions at the T7lac promoter on the pET vector to block transcription of the gene of interest by any T7 RNA polymerase that may be made due to leaky expression. Addition of IPTG blocks the inhibitory action of LacI, thereby inducing expression of T7 RNA polymerase and also removing LacI inhibition of the gene of interest.
Although the pET expression system is designed for high-level recombinant protein expression, the expression level can be reduced by decreasing the amount of IPTG supplied to host cells. This can be advantageous when expressing proteins with limited solubility. Additionally, the system is able to maintain the gene of interest in a transcriptionally silent state when T7 RNA polymerase is not present.
All custom pET vectors will be supplied in an E. coli strain designed to maximize plasmid integrity and lacking the T7 RNA polymerase gene (such as Stbl3). For recombinant protein production, we recommend transferring the vector to BL21(DE3) or HMS174(DE3) host bacteria strains, which carry chromosomal copies of the T7 RNA polymerase gene driven by the LacUV5 promoter. In cases when toxicity of the gene of interest is an issue in these expression host strains, the use of hosts carrying the pLysS or pLysE plasmids may be beneficial. These plasmids suppress basal expression of the gene of interest by producing T7 lysozyme, a T7 RNA polymerase inhibitor.
Strong expression: The T7 transcription and translation regulatory system allows for very high-level production of proteins of interest, in many cases close to 50% of total protein in the culture.
Tightly controlled expression: The expression of the gene of interest is generally very strongly repressed in the absence of added IPTG, and this “off” state is very robust for most genes of interest in most host strains.
Host requirements: Completed pET vectors should be maintained in an E. coli strain lacking the T7 RNA polymerase gene, and must be transferred to a separate host strain containing the T7 RNA polymerase gene before induction of protein expression.
Potential leaky expression in some hosts: Even in the absence of IPTG, there may be some low-level expression of T7 RNA polymerase from the LacUV5 promoter in some expression host strains, which could lead to bacterial toxicity for certain genes of interest in certain host strains.
T7 promoter: Drives high-level transcription of the gene of interest when T7 RNA polymerase is present. When placed immediately upstream of a LacO element, the entire cassette is known as the T7lac promoter.
LacO: Binding site for LacI. This element inhibits activity of the T7 promoter when LacI protein is present, preventing leaky expression of the gene of interest.
RBS: The ribosome-binding site and translation initiation element from T7 bacteriophage. This allows for efficient production of the protein of interest.
ORF: The open reading frame of your gene of interest is placed here.
T7 terminator: Signal sequence to terminate the transcript made from the gene of interest, preventing run-on transcription.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as well as the Rop gene exist in low copy numbers in E. coli.
Rop: Repressor of primer. It encodes a small protein that regulates plasmid copy number. The presence of the Rop protein, in combination of pBR322 origin of replication on the plasmid, results in low copy numbers of the plasmid.
LacI: The E. coli natural promoter and coding sequence for the lac repressor. In the absence of induction of the system (i.e. without IPTG), the LacI protein represses transcription of the gene of interest from the T7lac promoter, as well as transcription of T7 RNA polymerase from the LacUV5 promoter in host strains used for recombinant protein production.