Plasmid_Backbone
Screening

Part:pSB1A10

Designed by: Josh Michener, Jason Kelly   Group: iGEM2006_MIT   (2006-05-26)

Screening Plasmid v1.0

This plasmid can be used to do a rough characterization of the Input/Output curve for a PoPS-based device. Additionally, it can be used for screening of part or device libaries via FACS. More information can be found here. Note that this plasmid contains ccdb, so should be transformed into Part:BBa_V1005, or another compatible strain for initial preparation.

Schematic of Screening Plasmid 1.0: We are using the Pbad arabinose-inducible induction system as a tunable input Khlebnikov. GFP is a measure of input and RFP is a measure of output. A BioBrick® cloning site enables easy insertion of any BioBrick® part. RNase E sites create independence between the mRNA stability of the device being screened and the mRNA stability of the fluorescent proteins. In particular, we suspect mRFP1 contains internal RNaseE cut sites and have added a hairpin 5’ of the coding region to slow degradation by RNase E Smolke.


Note: The sequence below is incorrect because the backbone that the SP device was inserted into was psb1A2 rather than psb1A3, so there is a terminator (not annotated) in the sequence below at position 1044-1121 that isn't actually present in the DNA of psb1A10. Updating this will take some time because I need to change all the feature locations by hand - but the Vector NTI file is correct.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5242
    Illegal NheI site found at 4443
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 5248
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5242
    Illegal BglII site found at 89
    Illegal BamHI site found at 4382
    Illegal XhoI site found at 67
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 5242
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 5242
    Plasmid lacks a suffix.
    Illegal XbaI site found at 5257
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 674
    Illegal AgeI site found at 786
    Illegal AgeI site found at 4217
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2197
    Illegal BsaI.rc site found at 5118
    Illegal EcoRI site found at 5242
    Illegal XbaI site found at 5257
    Illegal SpeI site found at 2
    Illegal PstI site found at 16

References

<biblio>

  1. Bernard-Gene-1999 pmid=7926841
  2. Khlebnikov pmid=12080425
  3. Smolke pmid=12402322

</biblio>

[edit]
Categories
//classic/plasmid/measurement
//plasmid/measurement
//plasmidbackbone/copynumber/high
//plasmidbackbone/version/10
Parameters
chassis
copies100-300
mcsBioBrick
originpMB1
resistanceA