Plasmid
BB base ve

Part:BBa_I51020:Design

Designed by: Reshma Shetty   Group:   (2008-01-05)

BioBrick base vector (for constructing pSB**5 vectors)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1327
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1327
    Illegal NheI site found at 177
    Illegal NheI site found at 1126
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 1333
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1327
    Illegal BglII site found at 1783
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1327
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1327
    Illegal XbaI site found at 1342
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1671

Design Notes

In designing a BioBrick base vector that will serve as the basis for many new vectors, our priority was to ensure that the resulting vectors would fulfill the basic demands of the biological engineers who use them. We had five design requirements for our new BioBrick vectors.

  1. BioBrick standard vectors must be able to propagate any BioBrick standard biological part. To meet this requirement, the base vector must contain a complete BioBrick cloning site as specified in the original technical standardKnight-2003.
  2. BioBrick standard vectors must be easy to use for their most common purpose: assembly of BioBrick standard biological parts. To meet this requirement, we included BBa_P1016 within the BioBrick cloning site. BBa_P1016 encodes the positive selection marker ccdB. Positive selection markers prevent one of the most common problems during assembly of BioBrick parts: contamination of the ligation reaction with uncut plasmid DNASambrook-2001. Any cells transformed with the uncut plasmid DNA produce the lethal protein ccdB and dieBernard-Gene-1994 Bernard-Gene-1995 Bernard-Biotechniques-1996. Note, however, that the drawback of this solution is that inclusion of this part requires that users propagate both the base vector and any derived vectors in E. coli strains tolerant of ccdB expression such as DB3.1Bernard-J-Mol-Biol-1992 Miki-J-Mol-Biol-1992.
  3. BioBrick standard vectors should be easy to purify. To meet this requirement, we included a pUC19-derived origin (BBa_I50022) in addition to the ccdB selection marker within the BioBrick cloning siteVieira-Gene-1982 Norrander-Gene-1983 Yanisch-Perron-Gene-1985. The high copy origin encoded by BBa_I50022 means that both base vector DNA and any derived vector DNA are easily purified in large quantities, irrespective of whether the vector replication origin is low copy or notCabello-Nature-1976 Ioannou-Nat-Genet-1994. Cloning a BioBrick part into the BioBrick cloning site removes the high copy origin in the cloning site thereby restoring replication control to the vector origin.
  4. BioBrick standard vectors must stably propagate BioBrick parts. Previous work suggests that transcriptional isolation of the cloned DNA fragment can make those fragments more amenable to manipulationGentz-Proc-Natl-Acad-Sci-USA-1981 Stueber-EMBO-J-1982 Bowater-Nucleic-Acids-Res-1997 Godiska-2005. Thus, to meet this requirement, we included transcriptional terminators in both directions flanking the BioBrick cloning site (BBa_B0053, BBa_B0054, BBa_B0055 and BBa_B0062). By doing so, we sought to insulate the proper maintenance and propagation of the vector from the possibly disruptive function encoded by the BioBrick part cloned into the vector. We also flanked the BioBrick cloning site on both sides with translational stop codons in all six reading frames (BBa_B0042) to ensure translational insulation as well.
  5. BioBrick standard vectors should allow users to verify the length and sequence of the inserted BioBrick part. To meet this requirement and to support backwards compatibility with existing BioBrick parts and vectors, we incorporated the same primer annealing sites found in existing BioBrick vectors like pSB1A3-P1010 (BBa_G00100 and BBa_G00102). BBa_G00100 and BBa_G00102 are sufficiently distant from the BioBrick cloning site to ensure good quality sequence reads of any inserted part.

BBa_I51020 (BioBrick base vector) is composed of the BioBrick parts described. The BioBrick vector parts developed in this work confer upon the base vector, and therefore also any derived BioBrick standard vectors, the features that meet our design requirements.

To ensure that BBa_I51020 (BioBrick base vector) is easy to propagate and purify, we made BBa_I51020 itself a functional plasmid. We included an ampicillin resistance marker BBa_P1006 flanked by NheI restriction endonuclease sites (BBa_B0045) in the base vector BBa_I51020. BBa_P1006 is excised from BBa_I51020 upon construction of a new BioBrick standard vector. Thus, the combination of BBa_I50022 (high-copy replication origin) in the BioBrick cloning site and BBa_P1006 renders BBa_I51020 capable of autonomous plasmid replication for easy DNA propagation and purification.

Source

We use agarose gel electrophoresis of uncut plasmid DNA to determine that the DNA for BBa_I51001 (synthesized BioBrick base vector) was actually a fusion of two copies of BBa_I51001. To fix this issue, we performed a restriction digest of BBa_I51001 by mixing 0.5-1 μg DNA, 1X NEBuffer 2, 100 μg/ml Bovine Serum Albumin and 20 units EcoRI restriction enzyme in a 50 μL total volume. The reaction was incubated for 2 hours at 37°C and then heat-inactivated for 20 minutes at 80°C in a DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA). We then re-ligated the base vector by mixing 1 μL of a ten-fold dilution of the linearized BBa_I51001 DNA, 1X T4 DNA Ligase Reaction Buffer and 200 units T4 DNA Ligase in a 10 μL total volume. All reagents for these steps were purchased from New England Biolabs, Inc. in Ipswich, MA. We transformed the religated BioBrick base vector into E. coli strain DB3.1 via [http://openwetware.org/wiki/Knight:Electroporation electroporation] and plated the transformed cells on LB agar plates supplemented with 100 μg/mL ampicillin to obtain BBa_I51020. Correct construction of BBa_I51020 was verified by DNA sequencing by the MIT Biopolymers Laboratory.

References

<biblio>

  1. Bernard-J-Mol-Biol-1992 pmid=1324324
  2. Miki-J-Mol-Biol-1992 pmid=1316444
  3. Bernard-Gene-1994 pmid=7926841
  4. Bernard-Gene-1995 pmid=7557407
  5. Bernard-Biotechniques-1996 pmid=8862819
  6. Vieira-Gene-1982 pmid=6295879
  7. Norrander-Gene-1983 pmid=6323249
  8. Yanisch-Perron-Gene-1985 pmid=2985470
  9. Cabello-Nature-1976 pmid=765836
  10. Ioannou-Nat-Genet-1994 pmid=8136839
  11. Gentz-Proc-Natl-Acad-Sci-USA-1981 pmid=6946440
  12. Stueber-EMBO-J-1982 pmid=6327267
  13. Bowater-Nucleic-Acids-Res-1997 pmid=9207036

</biblio>