gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pBAD Bacterial Recombinant Protein Vector
The pBAD vector system is a reliable and controllable system for expressing recombinant proteins in bacteria. This system is based on the araBAD operon, which controls E. coli L-arabinose metabolism. The gene of interest is placed into the pBAD vector downstream of the araBAD promoter, which drives expression of the gene of interest in response to L-arabinose, and is inhibited by glucose. Precise control of expression levels makes this system ideal for producing problematic proteins, such as proteins with toxicity or insolubility issues.
For further information about this vector system, please refer to the papers below.
|J Bacteriol. 177:4121-30 (1995)||Development of pBAD vectors|
The gene of interest is cloned into the pBAD vector and maintained in growth medium lacking L-arabinose. This reduces plasmid instability that could result from the expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be induced by the addition of L-arabinose to the medium.
Our pBAD vectors contain the bidirectional araBAD promoter, which drives expression of both the gene of interest and the regulatory protein AraC. In the absence of L-arabinose, AraC dimerizes to form a loop in the promoter region, blocking transcription. When L-arabinose is added, AraC changes conformation and binds to alternate sites in the promoter, activating transcription.
Addition of glucose to the growth medium can further suppress basal expression due to a reduction in cellular cAMP levels. In glucose-free medium, cAMP levels are high, and a cAMP-CRP (catabolite activator protein) complex binds to the pBAD promoter. This association is required for promoter activity, so addition of glucose will robustly repress expression of the gene of interest. This is particularly useful when the gene of interest is toxic or inhibits bacterial growth.
All custom pBAD vectors will be supplied in an E. coli strain designed to maximize plasmid integrity (such as Stbl3). To express the protein of interest, the plasmid can be transferred to a host strain lacking genes for L-arabinose catabolism (such as TOP10). This allows efficient and stable gene activation without reduction in the L-arabinose concentration over time. For genes of interest that require tight suppression of basal expression by glucose, the LMG194 host strain should be used. This strain is capable of growth on minimal defined medium (RM medium), allowing additional repression of the araBAD promoter by glucose.
Very tightly controlled expression: Expression from pBAD vectors is more tightly controlled than from pET vectors. The expression of the gene of interest is at very low basal levels in the absence of L-arabinose, and is further repressed if glucose is present.
Strong induction: The araBAD operon system is highly inducible. It is possible to achieve >1000-fold induction upon removal of glucose and addition of L-arabinose.
Inexpensive induction: L-arabinose is inexpensive, making large-scale protein expression more economical.
Host requirements: Unlike pET vectors, pBAD can be maintained in the same E. coli strain that is used for induction, such as TOP10.
Sub-maximal expression: pBAD vectors are generally not capable of achieving the very high level of expression possible with pET vectors.
Metabolism of inducer: Wild-type E. coli strains can catabolize L-arabinose. When expressing the protein of interest, host strains that are mutant for L-arabinose catabolism (such as TOP10 or LMG194) should be used to avoid inconsistent expression due to depletion of L-arabinose in the medium over time.
araBAD promoter: Drives transcription of the gene of interest when L-arabinose is present and glucose is absent. This promoter also controls AraC expression.
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.
rrnB 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 exist in medium copy numbers in E. coli.
araC: Encodes the regulatory protein of the E. coli araBAD operon. AraC inhibits expression from the araBAD promoter in the absence of L-arabinose or the presence of glucose, and activates transcription in the presence of L-arabinose and the absence of glucose.