Plasmid_Backbone

Part:BBa_K187000:Design

Designed by: Team BioBytes, University of Alberta   Group: iGEM09_Alberta   (2009-10-15)

pAB Universal Plasmid for BioBytes Gene Assembly System

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 751
    Illegal XbaI site found at 793
    Illegal PstI site found at 838
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 751
    Illegal PstI site found at 838
    Illegal NotI site found at 757
    Illegal NotI site found at 862
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 751
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 751
    Illegal XbaI site found at 793
    Illegal PstI site found at 838
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 751
    Illegal XbaI site found at 793
    Illegal PstI site found at 838
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 1728
    Illegal SapI.rc site found at 773


Design Notes

The boundary sequences required for sticky end production for the BioBytes gene assembly system are acquired by inserting gene sequences destined to become bytes into either of the host plasmids pAB or pBA. Both are derivatives of pUC19 with the following key modifications:

1. Replacement of the pUC19 MCS with the MCS shown in figure 1.

2. Elimination of the Z alpha fragment

3. Elimination of the restriction sites Bts1 and BspQ1

Functional features include:

1. The ability to adapt biobricks to the Byte format using the Xba1 and Pst1 sites contained in the MCS.

2. The ability to adapt the in the ASKA collection (Kitagawa 2005) to the Byte format (4000+ E. coli K12 ORFs). ORFs are cloned into an expression vector, accompanied by a 6xHis tag at the N-terminus and a GFP fusion on the C-terminus. ASKA parts can be freed by digestion with SfiI, leaving noncomplementary 3 bp 3' sticky ends, which can be cloned directionally into the MCS after digestion with BstAPI. Since the ASKA parts are freed from the interior of the CDS, the MCS provides a start codon 10 bp downstream of the RBS, and a stop codon (TGA) downstream of the insertion. ORFs cloned from ASKA plasmids replace the N-terminal methionine of the predicted ORF with Met-Ala-Leu-Arg-Ala, and append Cys-Leu at the C-terminus.

3. Other potentially useful restriction sites have been included in the MCS as well as an RBS consensus positioned 8 bp upstream of the ATG used for ORF’s derived from 1. And 2.

4. The ability to select for insert-containing constructs through the linearization of parental contaminants by cleavage with the rare- cutters SwaI and PmeI contained in the MCS.

5. Two routes for the production of ~12 base overhangs by PCR using universal primers: A)PCR followed by removal of a primer-encoded uracil residue using BioLab’s USER TM system as per our RFC. Primer positions and sequences are indicated for both. B) PCR followed by nicking using the engineered enzymes Nb.BbvCI and Nb.BtsI. Note that only the USER method is compatible with RFC#47.

Source

Derivative of pUC19

References

Bitinaite, J. et al. USERTM friendly DNA engineering and cloning method by uracil excision. Nucl. Acids Res. 35:1992 (2007). doi:10.1093/nar/gkm041
Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. Kitagawa (2005)