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Plasmids

Gene And Cell Technologies provides a set of plasmids optimized for use with our SparkleCell bioreactors.  All our plasmids are provided under Open Source terms:  

  • Detailed use instructions are provided with each plasmid.
  • Fully validated with known yields and activity measures.
  • Annotated sequences are available.
  • Unlimited use licence -- these plasmids may be used for any legal purpose.* 

 

How are the pSparkle plasmids special?

In short, not very.  Any well-designed plasmid can work in SparkleCell, not just pSparkle.  The pSparkle plasmids are here to help you get the most out of your SparkleCell easily, but they are not mandatory. 

Basically the pSparkle plasmids just adhere to general good design practices for E. coli expression plasmids.  SparkleCell oxygenates cultures properly.  It makes the cells healthy, and thereby creates extra metabolic capacity to make your recombinant product.  The pSparkle plasmids give E. coli the correct set of instructions to take advantage of this extra capacity.  It helps ensure that there isn't any other, non-metabolic limitation limiting your product expression levels.  

For example, E. coli's ability to produce recombinant protein in the LB / shaker flask system is often topped out at around 50 mg / liter.  In SparkleCell it is common for that limit to go up to 300 mg / liter or more.  But if your protein is for some reason "difficult", and expresses at only 10 mg / liter in the shaker flask, then adding extra oxygen does not automatically help.  It might help, but that depends on what the problem is.  If your E. coli does not "try" to make any more of it, then giving it extra metabolic capacity might not be the right strategy.  You'll have to make it try first.  That's what the pSparkle backbone does: 

Features

Your gene of interest (GOI) is expressed from the tac promoter (green) as a fusion protein with E. coli cytoplasmic maltose binding protein (MBP).  

Tac_promoter:  This is one of E. coli's most highly expressing promoters.  It is a synthetic fusion promoter derived from the trp and lac operons, which has been designed in the 1980s for tight repression in combination with high inducibility [ref 1].  (You want to have tight repression during the growth phase of your E. coli, so that they are not unduly burdened by having to make protein during their growth phase).  The Tac promoter basically solved the problem of getting enough mRNA transcribed in E. coli for good.  There has been little progress in the field since, for lack of further opportunity to improve it.  If your gene is not expressing optimally, then the root of that problem is likely to be found elsewhere.

MBP:  This is E. coli's originally periplasmic maltose binding protein, expressed without secretion signal, and therefore re-targeted to the cytoplasm.  MBP is controlling the second major step in protein expression -- translation initiation at the ribosome.  Just like Tac causes a very high level of transcription initiation, MBP causes a high level of translation initiation.  It's one of the less well known facts about biology that translation initiation is not only controlled by Shine and Dalgarno's ribosome binding site [ref 2], but is also controlled to a large degree by the sequence and the structure of the actual amino acids that follow it, and that are being translated.  In this regard, MBP serves to crank the engine of translation into high gear.  It ensures that whatever follows is translated at extreme efficiency.  Even if your gene already works reasonably well in shaker flasks, the MBP fusion can help take full advantage of the extra metabolic capacity available in SparkleCell.

MBP has a few secondary benefits other than boosting the translation rate:  It acts as a chaperone that can sometimes dramatically improve the folding and biological activity of its fusion partner [ref 3].  For example we know from early development of our open source plasmid pSparkleTEV that the TEV protease would be 100% misfolded, inactive and expressed at a far lower level, if it wasn't expressed as an MBP fusion partner. 

MBP is also a built-in purification tag -- it binds with high efficiency to a maltodextrin or amylose column allowing one-step purification.  Elution is done under unmatched super-mild conditions, by simply adding 10 mM maltose.  It's possible to do so under aggressive protease inhibition schemes including high-dose EDTA.  On the flip-side, the purification resins tend to be expensive and suffer from low binding capacity.  (You might not be used to the idea that binding capacity is a problem -- just try the pSparkle / SparkleCell system:  You'll have your column's binding capacity maxed out so quickly!) That's why we've also included a central his-tag, between MBP and your gene of interest.  The his-tag (IMAC) resins can have up to 10x higher binding capacity than MBP tag resins.  But they're incompatible with protease inhibition schemes using EDTA.  We know experimentally that both the MBP and his-tags are suitable for purification with all cargo proteins tested so far. You can use either one of them, or both in series. 

The MBP and his-tags can be removed using the TEV protease cleavage site (Sequence "ENLYFQ//G") between the his-tag and your gene of interest.  TEV cleaves your fusion protein in two halves:  A his-tagged MBP fragment (which can be removed by either of those tags).  And your protein by itself, decorated only with a foreign "G" at its N-terminus.  This smallest of the amino acids is necessarily added due to its contribution to the TEV recognition site.  A his-and MBP tagged TEV protease can be obtained easily from pSparkleTEV.  pSparkleTEV does not have the cleavage site itself, so the tagged protease always stays in one piece and can be removed from a reaction with either tag. 

Ampicillin:  Ampicillin is the selective antibiotic used to maintain pSparkle inside E. coli.  E. coli transformed with pSparkle have the ability to destroy ampicililn and are resistant to ampicillin poisoning because of this gene.  When producing protein in SparkleCell, the culture can churn through a huge amount of ampicillin.  It will be necessary to replenish it multiple times according to the protocols shown in the manual, or by your own optimized schedule. 

rrnB is the transcription terminator that prevents wasting energy on excess transcription and improves mRNA stability.

pBR322, F1 and ROP are three DNA replication regulators acting in concert to maintain the plasmid in the cell at a precisely defined copy number and make sure it gets inherited to daughter cells.  Steady DNA copy number helps support the fidelity of the regulation of gene expression. 

LacI finally provides extra copies of the lac repressor.  The repressor prevents E. coli from having to make protein while it is growing up, so that it is not burdened, and does not face selective pressure against the best producers.  The cell does not have quite enough LacI molecules to achieve optimal repression of the lac regulator on this multi-copy plasmid.  That's why well designed expression plasmids usually include extra LacI.

 

Other Production Systems

Our "pSparkle" plasmids may work in systems other than SparkleCell, for example in shaker flasks.  However, once you run the numbers, you will find that our SparkleCell / pSparkle combination is by far the most effective and economical system for producing recombinant proteins in E. coli.  Furthermore, we can provide detailed expression protocols and technical support only for their use with SparkleCell.

 

References

[1] The tac promoter: a functional hybrid derived from the trp and lac promoters. de Boer HA, Comstock LJ, Vasser M. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21-5.

[2] Determinant of cistron specificity in bacterial ribosomes. Shine J, Dalgarno L. Nature. 1975 Mar 6;254(5495):34-8.

[3] Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Kapust RB, Waugh DS. Protein Sci. 1999 Aug;8(8):1668-74.

 

* Disclaimer:

Of course, we cannot speak for any third parties that may be able to restrict your use or impose fees, such as your employer, a patent owner, or the law.  All we're saying is that we don't impose any restrictions or fees of our own.  We are not responsible for any claims raised by third parties against you regarding your use of our products. 

 

 

 

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