What is the difference between vectors that contain a pUC origin and vectors that contain a pBR322 origin?

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Answer

Both pBR322 and pUC vectors have the same origin of replication. The locus that controls copy number is the "rop" locus; in pUC vectors, the rop locus is mutated to allow for much higher copy numbers. Therefore plasmids containing the pUC origin produce a high copy number of the plasmid. Most Invitrogen™ vectors have origins of replications that are pUC-derived and yield a high amount of DNA in E. coli. Plasmids derived from pBR322 will yield a lower plasmid copy number than plasmids derived from pUC. pBR322 gives about 15-20 copies per cell, while pUC gives about 500-700 copies.

Answer Id: E3247

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What strains of E. coli should be used with the pBAD inducible promoter system?

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Answer

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'.

Answer Id: E3254

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How should the pBAD promoter be induced when the cells are growing under maximal repression (i.e. in the presence of D-glucose)?

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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.

Answer Id: E3255

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What is the molecular weight of the L-arabinose included in the pBAD kits?

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Answer

The molecular weight of arabinose is 150.1 g/mol.

Answer Id: E3249

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Has Invitrogen™ mapped the exact transcriptional start site of the pBAD promoter?

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Answer

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

Answer Id: E3256

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

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Answer

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 (see figure below). 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.

Answer Id: E9713

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What concentration of tetracycline should be used for selecting the LMG194 strain?

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Answer

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

Answer Id: E3251

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

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Answer

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-.

Answer Id: E9717

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What is the purpose of the -35 and -10 consensus region, or sequence, that is found in prokaryotic promoters?

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Answer

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.

Answer Id: E3257

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

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Answer

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

Answer Id: E9714

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What media should be used to grow the LMG194 strain? Will LMG194 grow in M9 alone?

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Answer

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.

Answer Id: E3253

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

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Answer

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]).

Answer Id: E9712

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What is the best molar ratio of PCR product:vector to use for TOPO™ TA cloning? Is there an equation to calculate the quantity to use?

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Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO™ cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

Answer Id: E7648

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What protein yields should I expect to get with the pRSET expression vectors?

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Answer

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.

Answer Id: E3259

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

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Answer

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.

Answer Id: E9716

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