Featured Parts:CloningFacilitationTools
This page describes a set of plasmid variants to facilitate cloning. Basically, these elements are Biobrick parts that insert between the EcoRI and XbaI sites, or between SpeI and PstI site rather than usual Biobrick parts that insert between SpeI and PstI. All the elements described here can be used with standard EcoRI-XbaI-part-SpeI-PstI polylinker plasmids (pSB1A2, pSB1AK3, pSB2K3, pSB3C6, etc.) With respect to nomenclature, modifications outside of the XbaI/SpeI sites change the name of the vector. So, if you insert a cassette between the EcoRI and XbaI sites of plasmid pSB1AK3-b0015, the result would still be a *-b0015 plasmid, but a new name must replace the "*". At present, each basic plasmid part is receiving a standard biobrick part name, but this nomenclature may change later.
Plasmids for constructing basic Biobrick parts
One difficulty in the construction of basic biobrick parts is the absence of a phenotype without additional parts. To observe the activity of a part, one must therefore construct a composite part in addition to the basic part. This is particularly problematic when parts are identified from combinatorial libraries, where the basic part cannot be simply reconstructed from the materials used to make the composite part library. As a simplification to constructing basic parts, we have made a series of plasmids that retains the proper positioning of the XbaI and SpeI restriction sites flanking basic parts while allowing the introduction of the additional elements necessary to observe their activity.
Part J23006 is a pSB1A2-derived plasmid for constructing basic biobrick parts under the control of an external promoter. Insertion of parts such as a protein generator or a RNA key between the XbaI and SpeI (or) PstI sites places the part under the control of the part r0040 Tet promoter. The resulting plasmid retains the normal sequence context of a r0040.newpart composite with one point mutation changing the ACTAGA linker to TCTAGA. We have used this plasmid extensively to express RNA keys.
Plasmid J61002, like J23006, is a pSB1A2-derived plasmid for constructing basic biobrick promoter parts. When new promoters or libraries of promoters are placed within the XbaI and SpeI sites, they control the expression of RFP while retaining their ability to be used in Biobrick standard assembly. This plasmid was used for the construction of our constitutive promoter library.
Plasmids J23018 and J23019 allow the construction of open reading frame basic parts while allowing their constitutive expression on a colE1 or p15A origin of replication, respectively.
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