Measurement

Part:BBa_J100091

Designed by: Malcolm Campbell and Todd Eckdahl   Group: Campbell_M_Lab   (2012-08-20)

For Testing New Promoters via Golden Gate Assembly

J100091 (also called pClone Basic) was built by Todd Eckdahl and Malcolm Campbell. This part allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see Part:BBa_J119022 as an example, or the picture below). Successful GGA assembly replaces the double terminator in J100091 with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. J100091 and its sister part Part:BBa_J119044 incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.

Below is a picture of the portion that pops out when digested with Bsa I. TT represents the transcriptional terminator Part:BBa_B0014. J100091 is designed to be used for Golden Gate Assembly to swap out the TT for a promoter of your choosing. This can be done by simply mixing J100091 with oligos that self-assemble into dsDNA with compatible sticky ends. J100091 and its sister part Part:BBa_J119044 are ideal for undergraduates in a teaching lab to discover and characterize new promoters for use in synthetic biology.

See the full GGA protocol online.

J100028.png


Sequence and Features
J100091 contains BBa biobrick prefix + 4 base overhang + BsaI site cutting to the left + transcriptional terminator Part:BBa_B0014 + BsaI site that cuts to the right + 4 base overhang + BD18 bicistronic RBS for more reliable translation (Part:BBa_J119024) + mRFP Part:BBa_E1010 + BBa biobrick suffix.


Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 906
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal NotI site found at 7
    Illegal NotI site found at 914
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 907
    Illegal PstI site found at 921
    Illegal AgeI site found at 779
    Illegal AgeI site found at 891
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 129
    Illegal BsaI.rc site found at 28
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 907
    Illegal PstI site found at 921


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Categories
Parameters
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