Plasmid_Backbone
pSB1A7

Part:pSB1A7:Design

Designed by: Karmella Haynes   Group: iGEM06_Davidson   (2006-10-19)
Revision as of 19:59, 21 October 2006 by Rshetty (Talk | contribs)

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Design Notes

Purpose
Read-through transcription is extremely problematic when the usefulness of a device depends upon an off state. This cloning vector was constructed to insulate BioBrick parts from read-through transcription coming from the backbone.

The following parts show expression in cloning vectors pSB1A2 and pSB1A3, even in the absence of a promoter (or where the promoter is not in the proper orientation)

  • BBa_S03562 - Promoterless tetracycline resistance coding region (forward orientation) with a ribosomal binding site (RBS-TetF) is expressed (tet resistant) in pSB1A2 and pSB1A3
  • BBa_S03532 - Promoterless tetracycline resistance coding region (backward orientation) with a backwards ribosomal binding site (TetB-RBS) is expressed (tet resistant) in pSB1A2 and pSB1A3
  • BBa_J3106 - pBad promoter in the forward orientation followed by TetB-RBS in the reverse orientation (pBad-hixC-TetB-RBS-hixC-TT-RE) is expressed (tet resistant) in pSB1A2
  • BBa_J44004 - pBad promoter in the reverse orientation followed by RBS-TetF (hixC-pBadrev-hixC-RBS-TetF)

These observations suggest that there is forward and reverse read-through coming from the backbone of the carrier vector into the BioBrick parts.


Double Forward and Backwards Terminator Assembly
The double forward terminator BBa_B0015 successfully blocks RBS-TetF from read through in pSB1A2, resulting in th loss of tet resistance (see S03595 Part Design for details).

The double terminators used to build pSB1A7 were assembled from smaller overlapping ssDNA oligos. These oligos were designed using the [http://gcat.davidson.edu/IGEM06/oligo.html Do It Yourself Gene Assembly Program] created by Davidson College.

  • Left Double Terminator (forward): The BBa_B0015 sequence was modified as follows: a 'G' was added to the 5' end (to serve as an EcoRI site mutation); a sequence containing the EcoRI and NotI sites of the BioBrick prefix (5'-GCA GAATTC GCGGCCGC T T-3') was added to the 3' end. This sequence was entered into the gene assembly program and ssDNA oligo sequences were obtained. A 5'-AATG-3' extension was added to the 5' end of first plus strand oligo to produce an EcoRI sticky end. A 5'-CTAG-3' extension was added to the 5' end of the final minus strand oligo to produce an XbaI sticky end. Equimolar amounts of oligos were mixed and annealed. This final product was used directly in a subsequent ligation reaction.
  • Right Double Terminator (reverse): The BBa_B0015 complimentary sequence was modified as follows: a 'G' was added to the 3' end (to serve as a PstI site mutation); a sequence containing the NotI and PstI sites of the BioBrick suffix (5'-T A GCGGCCG CTGCAG GCT) was added to the 5' end. This sequence was entered into the gene assembly program and ssDNA oligo sequences were obtained. A 5'-CTAG-3' extension was added to the 5' end of the first plus strand oligo to produce an SpeI 5' sticky end. A 5'-TGCA-3' extension was added to the 3' end of the final plus strand oligo to produce a PstI sticky end. Equimolar amounts of oligos were mixed and annealed. This final product was used directly in a subsequent ligation reaction.


Assembly and Functional Confirmation of pSB1A7

  • The right double terminator (reverse) was inserted to the right of TetB-RBSrev (BBa_S03532) in pSB1A3 at SpeI / PstI (!). The insertion of the double terminator resulted in a loss of tet resistance, thus functionality of the reverse BBa_B0015 was confirmed.
  • The left double terminator (forward) was inserted to the left of Tet-RBSrev in pSB1A3 at EcoRI / XbaI (!). The resulting cloning vector carries an intact BioBrick MCS while the EcoRI and PstI sites outside the double terminators are mutated so that the terminators can remain intact during cloning.
  • Tet-RBS was excised with EcoRI / PstI to produce an empty vector. RBS-TetF (BBa_S03562) was inserted into the empty vector. Functionality of the forward double terminator was confirmed by loss of tet resistance.
  • Cloning vector pSB1A4 is maintained carrying RBS-TetF.

! Note: Only a single clone was maintained for subsequent steps to ensure that polymorphisms that may have been introduced during oligo synthesis would not be propagated.


Caveats
This cloning vector is not compatible with parts containing he double forward terminator BBa_B0015. All attempts to insert various parts containing BBa_B0015 into pSB1A7 have lead to cloning failure in JM109. We suspect that parts containing an extra copy of the double terminator overload JM109 with inverted repeats. We plan on creating another version using the same approach, but different terminators.

Source

Based upon pSB1A3. Includes two double terminators assembled from DNA oligos based upon the sequence of BBa_B0015.

References