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| ==Design== | | ==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. |
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| 5' -- VF2 -- plasmid barcode -- BBa_B0055 -- BB prefix -- BBa_P1010 -- ?pUC19 backbone? -- BBsuffix -- BBa_B0054 -- VR -- BBa_I50000 -- antibiotic resistance cassette -- 3'
| | 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. |
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| ===Proposed features===
| | 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. |
| *F plasmid backbone
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| *positive selection marker (i.e. ''ccdB'' or ''sacB'') in between BioBricks restrictions sites to facilitate cloning
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| *some mechanism for putting the vector at high copy for purification purposes
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| *strong terminators flanking the BioBricks insertion site
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| *no loxP or cos insertion sites or Tn7 attachment sites?
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| **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?
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| *no blue-white screening?
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| **inclusion of a ''lacZα'' fragment would restrict its use as a part
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| *multiple versions with different antibiotic resistance markers
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| *no selection system for mammalian cells
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| *VF2 and VR sites
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| *Unique tag near but outside the cloning sites for identification during sequencing. (from Randy)
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| *orient the antibiotic resistance cassette on the reverse strand from the BioBricks insertion site
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| *include a topoisomerase binding site outside of the BioBricks cloning site for TA cloning.
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| **remove all topoisomerase binding sites in the vector
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| | ==Fabrication== |
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| | *'''[[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. |
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| | ==Notes== |
| ===Drawbacks=== | | ===Drawbacks=== |
| *Can only be used in F<sup>-</sup> strains | | *Can only be used in F<sup>-</sup> strains |
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| *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. |
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| ==Planning== | | ==Relevant pages== |
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| ===Current status===
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| The following parts have been designed
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6307 BBa_I50000]: F plasmid backbone with BioBricks restriction sites removed
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6336 BBa_I50010]: oriV origin which requires TrfA protein to be functional.
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| *[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=6400 BBa_I50020]: high copy origin from pSB1A3
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6306 BBa_B0055]: upstream flanking terminator
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6305 BBa_B0054]: downstream flanking terminator
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| The following parts have been designed, fabricated and tested
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6370 BBa_P1001]: cassette providing tetracycline resistance (from [[Austin Che]])
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6369 BBa_P1000]: cassette providing chloramphenicol resistance (from [[Austin Che]])
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| *[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=4941 BBa_I1000] or [http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=6396 BBa_P1010]: ccd operon in BioBricks format (from Leon Chan)
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| ===To do list===
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| *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.
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| **I have TetR and CmR BioBricked using Tom's primers. --[[User:Austin|Austin]] 18:26, 3 Dec 2005 (EST)
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| *Design unique identifiers for vectors.
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| *Design a pUC19 backbone for possible use in the MCS so that the plasmid can be purified at high copy
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| *Check for topoisomerase binding sites in the vector parts and remove them.
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| ===To be decided===
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| *Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
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| **Can we get a price break for synthesizing multiple plasmid variants?
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| **How many assemblies would we need to do?
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| **Is there a hybrid approach? Could we PCR the F plasmid backbone since its long and then have the collection of smaller parts (that would otherwise involve several assemblies) synthesized? Maybe a partial synthesis approach would help get around the issue of constructing a BioBricks insertion site?--[[User:Bcanton|BC]]
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| ***PCR'ing the F plasmid backbone is not very practical since there are several BioBricks sites in the backbone each of which would need to be individually mutated out. It is unlikely that there is anyone who is willing to do this much work. Therefore, the current plan is to synthesize the backbone. --[[Reshma Shetty | RS]]
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| *If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
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| **Blunt-end ligation?
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| **Other restriction enzyme sites?
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| **PCR
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| *Use a secondary, inducible copy number origin or insert the pUC backbone inside of the BioBricks cloning site to facilitate prepping of the vector?
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| **Inducible copy number origin
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| ***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.
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| ***The plasmid should operate at single copy in most other strains.
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| ***Systems containing the arabinose promoter would not be able to be induced to higher copy in the special strain without also affecting system behavior.
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| ***This option has the advantage that parts can be prepped from the F plasmid.
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| ***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.
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| ****I must not be understanding this, isn't DB3.1 resistant to "ccdB" and hence you wouldn't be using DB3.1 for selection anyway?
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| *****I was referring to the fact that prepping the plasmid for use would be difficult. I envision using this plasmid not to assemble BioBrick parts but as the vector in which characterization takes place. Therefore, I assume we would more often want to prep the vector (which contains ''ccdB'') than a vector with a part in it. Thus, in order to easily prep large amounts of vector, we would need a host strain that can tolerate ''ccdB'' and has the ''traF'' copy up mutation which likely does not exist (and therefore would need to be constructed). I am not convinced that we will want to prep parts in the F plasmid so frequently that it is worth the effort of constructing such a strain. --[[Reshma Shetty | RS]]
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| **pUC19 origin in the BioBricks insertion site
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| ***simply inserting a pUC backbone into the BioBricks insertion site enables the plasmid to be prepped easily and does not introduce any incompatibility issues.
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| ***allows the plasmid to be prepped at very high copy
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| ***parts in the F plasmid cannot be easily prepped
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| ****So this means we'd have to TempliPhi in order to sequence? The TempliPhi [http://www1.amershambiosciences.com/aptrix/upp01077.nsf/Content/autodna_templiphi_intro website] seems to think this is reliable enough to work--[[User:Bcanton|BC]]
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| ****Sequencing could be done by either sequencing a PCR product or via Templiphi. You can prep F plasmid from cells, it is just not quite as simple and quick as a miniprep since you get much lower yields. --[[Reshma Shetty | RS]]
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| *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?
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| **PCR
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| **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.
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| ===To be determined===
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| *Are we sure that F plasmids are really at 1-2 copies per cell? Why was pSB2K3-1 measured to be higher than that?
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| *What parts of the F plasmid are responsible for integration onto the genome? Can we omit them?
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| **cos and/or loxP sites are generally used for integration in the genome. Currently, I have no plans to include them in this vector.
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| *Many of the existing BACs only seem to have a partial ''sopC'' CDS, do we want the rest?
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| **pSMART VC vector appears to have a more complete ''sopC'' region. This may lead to tighter control of copy number.
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| *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)
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| **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]]
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| ** Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. --[[Reshma Shetty | RS]]
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| ==Notes==
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| See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]]. | | See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]]. |
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| [[Category:Project]] | | [[Category:Project]] |
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| | ==References== |
| | <biblio> |
| | #Jones-BiotechnolBioeng-1998 pmid=10099385 |
| | #Metcalf-Gene-1994 pmid=8125283 |
| | </biblio> |