Invitrogen™ pBAD/gIII Kit

Catalog No.	Quantity
V45001	1 kit

Catalog No. V45001 Supplier Invitrogen™ Supplier No. V45001

Description

Includes

T20μg each of pBAD/gIII A, B and C, 20μg of pBAD/gIII/calmodulin, 1mL 20% L-arabinose, LMG194 and TOP10 E. coli stabs.

Express recombinant proteins under the tight regulation of the araBAD promoter

• To simplify purification, vector pBAD/gIII encodes a leader peptide which directs recombinant protein into the periplasmic space
• Features araBAD promoter for tightly regulated expression
• Translation initiation signals for optimized E. coliexpression
• Leader peptide from bacteriophage fd gene III protein (gIII) for secreted expression
• C-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin
• C-terminal c-myc epitope for detection and analysis with an Anti-myc Antibody
• A set of three vectors is provided (A, B, and C): Each has the C-terminal tag in a different reading frame relative to the multiple cloning site to simplify in-frame cloning of the gene

Bacterial Expression, Protein Expression, Proteins, Expression, Isolation and Analysis

Specifications

• Constitutive or Inducible System: Inducible
• Inducing Agent: Arabinose
• Promoter: araBAD
• Product Type: Bacterial Expression Vector
• Selection Agent (Eukaryotic): None
• Content And Storage: The pBAD/gIII Kit includes 20 μg each of pBAD/gIII A, B, & C, 20 μg of pBAD/gIII/calmodulin, 1 ml 20% L-arabinose, LMG194 and TOP10 E. coli stabs. Store stabs at 2 - 8°C. Store all other components at -20°C. All components are guaranteed stable for 6 months when properly stored.
• Antibiotic Resistance Bacterial: Ampicillin (AmpR)
• Protein Tag: c-Myc Epitope Tag
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 1 kit
• Vector: pBAD

Frequently Asked Questions (FAQs)

How much L-arabinose should I use to induce expression with the pBAD expression system?

While the amount of L-arabinose can vary depending on your expression experiment, we suggest performing a pilot expression experiment with varying amounts of L-arabinose from 0.00002% to 0.2%.

Should I use TOP10 cells or the LMG194 E. coli strain you offer for expression with my pBAD system?

Top10

Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.

Disdvantages:
- This strain is not protease-deficient. Therefore, the protein may be degraded.

LMG194

Advantages:
- Grows well in minimal media, except M9.
-Have to transform the plasmid into the cells just for expression.
-RM medium with glucose to ensure low basal level of protein.

Disadvantages:
- Not protease-deficient. Therefore, the protein may be degraded.
- The glycerol stock may not be stable because this cell strain is not recA- or endA-.

What competent cells do you recommend I use for expression with my pBAD expression system?

We recommend using a competent cell strain that is araBADC- and araEFGH+, allowing transportation of L-arabinose, but not metabolizing it. This is important for expression studies, as the level of L-arabinose will be constant inside the cell and will not decrease over time. We offer our TOP10 competent cells, or our LMG194 E. coli strain.

Can you tell me the sequence of primers that can be used in pBAD vectors to sequence my gene of interest?

The pBAD vectors contain a forward and reverse pBAD primer flanking the gene of interest. The sequences are as follows:

pBAD forward primer: 5'-ATGCCATAGCATTTTTATCC-3'

pBAD reverse primer: 5'-GATTTAATCTGTATCAGG-3'

Do you have a list of the cap colors for the pBAD vectors?

Here are the cap colors:

- pBAD/His A: Red

- pBAD/His B: Orange

- pBAD/His C: Yellow

- pBAD/His LacZ: Green

- pBad/gIII A: Yellow

- pBad/gIII B: Green

- pBad/gIII C: Blue

- pBAD/gIII/calmodulin: Purple

How does the regulation of the araBAD (pBAD) promoter work?

The araBAD promoter (pBAD) used to control expression of T7 RNA polymerase in BL21-AI is both positively and negatively regulated by the product of the araC gene (Ogden et al., 1980; Schleif, 1992). AraC is a transcriptional regulator that forms a complex with L-arabinose. In the absence of L-arabinose, the AraC dimer contacts the O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop. For maximum transcriptional activation, two events are required.

- L-arabinose binds to AraC and causes the protein to release the O2 site and bind the I2 site that is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin.
- The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2.

Please note, basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is decreased.

What arabinose should I use for induction of my pBAD expression system?

We recommend using L-arabinose to regulate expression of your gene when using the pBAD expression system. In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD (Lee, 1980 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1980+pbad]; Lee et al., 1987 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1987+arbinose]).

What are the benefits of the pBAD expression system?

The pBAD expression system allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli. The system helps to produce proteins at a level just below the threshold at which they become insoluble. The regulatory elements of the E. coli arabinose operon are used in the pBAD vectors to precisely modulate heterologous expression levels, allowing optimization of the yields of the protein of interest. The regulatory protein, AraC, is provided on the pBAD vector backbone, allowing for regulation of pBAD.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

What is the purpose of the LMG194 strain provided in the pBAD Kits?

The LMG194 strain is provided to allow for growth of your plasmid construct under conditions of maximal repression. This strain is capable of growth on minimal media that includes glucose as the sole carbon source. Glucose provides maximal repression of the pBAD promoter. Use of the LMG194 strain is not absolutely necessary since the TOP10 strain may also be used for expression.

Note: all suitable strains must have mutations in the ara gene locus to prevent the breakdown of arabinose when it is added to the medium.

I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is a Shine-Dalgarno sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA.

For Research Use Only. Not for use in diagnostic procedures.