What is the purpose of the RBS sequence found in prokaryotic vectors?

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Answer

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.

Answer Id: E3258

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

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

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

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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'

Answer Id: E9715

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

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

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

Answer Id: E3251

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Which form of arabinose should be used for induction in the pBAD Expression System, L- or D-arabinose?

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The chirality of arabinose used for induction is very important. L-arabinose works great, but D-arabinose doesn't induce at all. L-arabinose is available from Sigma (catalog# A3256).

Answer Id: E3248

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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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Answer

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

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

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Answer

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.

Answer Id: E3252

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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 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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