Invitrogen™ pBAD/Myc-His Kit

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.

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]).

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.

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.

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.

We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.

The RBS, which stands for Ribosomal Binding Site, is required for translational initiation in E. coli, and consists primarily of purines. The consensus core region, or Shine-Dalgarno sequence (AGGAGG) is frequently located 8-12 base pairs upstream of the initiating codon AUG. This sequence forms base pairs with a complementary sequence located at the 3' end of the 16S rRNA molecule of the ribosome and assists in the recognition of the proper AUG of translation initiation.

In prokaryotes, such as E. coli, the promoter consists of two short regulatory sequences located 10 and 35 nucleotides upstream of the gene, respectively. The sequence located at -10 is called the Pribnow box (consensus sequence: TATAAT) and is absolutely essential for the transcription initiation. The sequence at -35 (consensus sequence: TTGACA) facilitates high transcription rate. Both regions are recognized by the sigma subunit of RNA polymerase, and instruct the holoenzyme where to start transcription. Once polymerization begins, the sigma subunit dissociates, and the core enzyme continues to transcribe the DNA template.

No, the exact start site of the pBAD promoter has not been determined experimentally.

If the construct is growing under maximal repression (i.e. in LMG194 with D-glucose in RM media), the cells should be harvested and resuspended in RM medium containing 0.2% glycerol and the appropriate concentration of arabinose (determined empirically). If cells are growing in LB medium, then arabinose may be added directly to the medium. Note that TOP10 cells will not grow in RM medium.

Any E. coli strain that is araBADC- and araEFGH+ is suitable for use with the pBAD promoter. Suitable strains that can be used include TOP10, TOP10F', and DH10B. Cells that are not araBADC- and therefore cannot be used with pBAD constructs, include DH5alpha, OmniMAX, Mach1, BL21(DE3) and INValphaF'.

LMG194 will grow in LB and RM medium that contains M9 salts. LMG194 will not grow in M9 salts alone. RM medium is used to ensure low basal expression levels from the pBAD promoter.

Please note that we do not support the use of LMG194 cells for any applications other than with the pBAD expression system.

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.

The LMG194 strain can be selected on LB plates containing 25 ug/mL tetracycline.

The molecular weight of arabinose is 150.1 g/mol.

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.

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

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

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.