Synthetic Biology:Vectors/Single copy plasmid: Difference between revisions

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See [[Vectors]] for information on [[Vector nomenclature | vector nomenclature]], [[Available vectors | existing vectors]] and [[Vectors to be constructed | vectors that we would like constructed]].
==Goal==
Design and fabricate a single copy vector in which BioBricks devices can be characterized.  To date most characterization work has been done in low or high copy vectors which have several issues including
# Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.
# At high copy, devices place a high metabolic load on the cell thereby altering host physiology and observed device behavior.
The proposed solution to these two problems is to characterize devices at single copy in the cell.  Obviously, such a vector will vary between 1 and 2 copies per cell over the cell cycle but nevertheless will hopefully present an improvement over the current situation.  The advantage of using a single copy plasmid rather than simply integrating the device into the genome is that a separate plasmid offers some isolation from the host and makes moving the device between different host strains slightly easier.


==Construction of single copy BioBricks vector==
==Design==
The F plasmid origin needs to be designed.  The complete F plasmid with partitioning genes in ~10kb in length.  It contains several BioBricks restriction sites in both coding and noncoding regions.


See notes on [[Bacterial artificial chromosomes | bacterial artificial chromosomes]].
Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the [[Synthetic Biology:Vectors/pSB**5 design|vector scaffold]] to generate a new single copy BioBricks plasmid.


===Design===
Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site).  The R6K origin is a conditional origin.  It only works in the presence of the trans-acting protein Π (encoded by pir) for replication.  R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts.


5' -- VF2 -- plasmid barcode -- BBa_B0055 -- BB prefix -- BBa_I1000 -- ?pUC19 backbone? -- BBsuffix -- BBa_B0054 -- VR -- BBa_I50000 -- antibiotic resistance cassette -- 3'
==Fabrication==


====Proposed features====
*'''[[User:Rshetty|Reshma]] 15:27, 27 February 2007 (EST)''': Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method.
*F plasmid backbone
*positive selection marker (i.e. ''ccdB'' or ''sacB'') in between BioBricks restrictions sites to facilitate cloning
*some mechanism for putting the vector at high copy for purification purposes
*strong terminators flanking the BioBricks insertion site
*no loxP or cos insertion sites or Tn7 attachment sites? 
**I can't think of an obvious use of these sites unless we want to build in the capability for integrating onto the genome.  However, wouldn't it make more sense to build in recombination capabilities onto a higher copy number vector than this?
*no blue-white screening? 
**inclusion of a ''lacZα'' fragment would restrict its use as a part
*multiple versions with different antibiotic resistance markers
*no selection system for mammalian cells
*VF2 and VR sites
*Unique tag near but outside the cloning sites for identification during sequencing. (from Randy)
*orient the antibiotic resistance cassette on the reverse strand from the BioBricks insertion site


====Drawbacks====
==Notes==
===Drawbacks===
*Can only be used in F<sup>-</sup> strains
*Can only be used in F<sup>-</sup> strains
*Should likely be used in ''recA<sup>-</sup>'' strains to avoid integration onto the genome and ensure plasmid stability.
*Should likely be used in ''recA<sup>-</sup>'' strains to avoid integration onto the genome and ensure plasmid stability.
*It is unclear whether this vector would truly be operating at single copy.  If it is not, perhaps it is easier to stick with the pSB2* plasmids.
*It is unclear whether this vector would truly be operating at single copy.  If it is not, perhaps it is easier to stick with the pSB2* plasmids.


===Planning===
==Relevant pages==


====Current status====
See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]].
The following parts have been designed
*[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=4941 BBa_I1000]: ccd operon in BioBricks format
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6307 BBa_I50000]: F plasmid backbone with BioBricks sites removed
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6336 BBa_I50010]: oriV origin which requires TrfA protein to be functional.
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6306 BBa_B0055]: upstream flanking terminator
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6305 BBa_B0054]: downstream flanking terminator


====To do list====
See notes on [[Bacterial artificial chromosomes | bacterial artificial chromosomes]].
*One of the things needed for this project is BioBricked antibiotic resistance cassettes.  This is also a prerequisite for the [[Standard E. coli Strain for BioBricks|standard strain project]].  Tom has ordered primers and is planning on cloning several resistance cassettes.
**I have TetR and CmR BioBricked using Tom's primers. --[[User:Austin|Austin]] 18:26, 3 Dec 2005 (EST)
*Design unique identifiers for vectors.
*Design a pUC19 backbone for possible use in the MCS so that the plasmid can be purified at high copy


====To be decided====
See [[Synthetic Biology:Vectors]] for information on [[Synthetic Biology:Vectors/Nomenclature | vector nomenclature]], [[Synthetic Biology:Vectors/Inventory | existing vectors]] and [[Synthetic Biology:Vectors/Wishlist | vectors that we would like constructed]].
*Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
**Can we get a price break for synthesizing multiple plasmid variants
**How many assemblies would we need to do?
*If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
**Blunt-end ligation?
**Other restriction enzyme sites?
**PCR
*Use an secondary, inducible copy number origin or insert the pUC backbone inside of the BioBricks cloning site to facilitate prepping of the vector?
**Inducible copy number origin
***The most common system for inducible copy number F-based plasmid requires a special strain (a copy up mutant of ''traF'' under the control of an arabinose inducible promoter) for inducible behavior. 
***The plasmid should operate at single copy in most other strains. 
***Systems containing the arabinose promoter would not be able to be induced to higher copy in the special strain without also affecting system behavior. 
***This option has the advantage that parts can be prepped from the F plasmid.
***Could not easily make use of ''ccdB'' as a selection marker because DB3.1 does not express the ''traF'' gene necessary for expression at high copy.  ''sacB'' is an alternative but requires sucrose in the media.
**pUC19 origin in the BioBricks insertion site
***simply inserting a pUC backbone into the BioBricks insertion site enables the plasmid to be prepped easily and does not introduce any incompatibility issues.
***allows the plasmid to be prepped at very high copy
***parts in the F plasmid cannot be easily prepped
*What is the best way to assemble a pUC backbone with the F plasmid backbone such that the pUC backbone is flanked by BioBricks sites?
**PCR
**Use special restriction sites for vector construction (Austin's idea).  Expanding on this, we could define a new idempotent assembly standard for exclusive use for vector components.


====To be determined====
[[Vectors]] has a lot of general information on vectors.
*Are we sure that F plasmids are really at 1-2 copies per cell?  Why was pSB2K3-1 measured to be higher than that?
*What parts of the F plasmid are responsible for integration onto the genome?  Can we omit them?
**cos and/or loxP sites are generally used for integration in the genome.  Currently, I have no plans to include them in this vector.
*Many of the existing BACs only seem to have a partial ''sopC'' CDS, do we want the rest?
**pSMART VC vector appears to have a more complete ''sopC'' region.  This may lead to tighter control of copy number.
*A set of orthogonal single copy replication origins to allow multiple vectors to be maintained in a cell.  Can we have a set of vectors with F and P1 origins?--[[User:Bcanton|BC]] 17:36, 31 Oct 2005 (EST)
**Not sure this is possible.  I believe the P1 origins use the par set of genes to maintain single copy whereas the F origins use the sop set of genes.  The two sets are pretty homologous to eachother and therefore likely incompatible.  I need to check on this more.  --[[Reshma Shetty | RS]]
** Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. --[[Reshma Shetty | RS]]


==BioBrick plasmid parts==
[[Category:Project]]


*[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=4941 BBa_I1000]: ccd operon in BioBricks format
==References==
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6307 BBa_I50000]: F plasmid backbone with BioBricks sites removed
<biblio>
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6336 BBa_I50010]: oriV origin which requires TrfA protein to be functional.
#Jones-BiotechnolBioeng-1998 pmid=10099385
*antibiotic resistance cassettes (from Tom)
#Metcalf-Gene-1994 pmid=8125283
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6306 BBa_B0055]: upstream flanking terminator
</biblio>
*[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6305 BBa_B0054]: downstream flanking terminator
*pUC19 high copy backbone
*unique identifier for the vector
 
[[Category:Project]]

Latest revision as of 13:27, 27 February 2007

Goal

Design and fabricate a single copy vector in which BioBricks devices can be characterized. To date most characterization work has been done in low or high copy vectors which have several issues including

  1. Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.
  2. At high copy, devices place a high metabolic load on the cell thereby altering host physiology and observed device behavior.

The proposed solution to these two problems is to characterize devices at single copy in the cell. Obviously, such a vector will vary between 1 and 2 copies per cell over the cell cycle but nevertheless will hopefully present an improvement over the current situation. The advantage of using a single copy plasmid rather than simply integrating the device into the genome is that a separate plasmid offers some isolation from the host and makes moving the device between different host strains slightly easier.

Design

The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions.

Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the vector scaffold to generate a new single copy BioBricks plasmid.

Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts.

Fabrication

  • Reshma 15:27, 27 February 2007 (EST): Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method.

Notes

Drawbacks

  • Can only be used in F- strains
  • Should likely be used in recA- strains to avoid integration onto the genome and ensure plasmid stability.
  • It is unclear whether this vector would truly be operating at single copy. If it is not, perhaps it is easier to stick with the pSB2* plasmids.

Relevant pages

See the list of parts for plasmid engineering.

See notes on bacterial artificial chromosomes.

See Synthetic Biology:Vectors for information on vector nomenclature, existing vectors and vectors that we would like constructed.

Vectors has a lot of general information on vectors.

References

  1. Jones KL and Keasling JD. Construction and characterization of F plasmid-based expression vectors. Biotechnol Bioeng. 1998 Sep 20;59(6):659-65. PubMed ID:10099385 | HubMed [Jones-BiotechnolBioeng-1998]
  2. Metcalf WW, Jiang W, and Wanner BL. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene. 1994 Jan 28;138(1-2):1-7. DOI:10.1016/0378-1119(94)90776-5 | PubMed ID:8125283 | HubMed [Metcalf-Gene-1994]

All Medline abstracts: PubMed | HubMed

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