NanoBio: Commonly Used Plasmids
3 plasmid assembly vectors: pSB1AC3, pSB1AK3, pSB1AT3
- Here are the annotated sequences of the pSB1AC3, pSB1AK3, pSB1AT3.
- These plasmids contain the ccdB gene, which is a toxin to DNA gyrase, so they must be propagated in TOP10 ccdB Survival Cells (Invitrogen catalog # C7510-03). These cells contain the ccdA gene, an antidote to ccdB, integrated in the bacterial genome.
- These plasmids are lower copy than the V0100/V0120 plasmids, so that ~10 ug of plasmid can be mini-prepped from a 5 mL overnight culture.
- Note: Unlike ampillicin selection, transformants must be grown in SOC for ~1 hr. before being plated unto chlorenphenicol, kanamicin, or tetracycline-containing media.
BioFusion Vectors: V0120, V0100 and V0002
- We commonly use two Biobrick vectors: V0120 (same as V0100) and V0002. The V0120 vector preferred over the V0002 vector because V0120 is a high-copy plasmid (~300 ng/uL from a mini-prep of a 5 mL culture) while V0002 is not. The V0002 plasmid is most frequently found in older parts.
- The vector backbone of V0100 & V0120 are identical. The only difference is that the V0120 vector contains a part in between the Biobrick ends, while the V0100 does not.
- The V0120 vector contains a part composed of the coding regions for the ccdB death gene and chloramphenicol resistance. Because of this, the V0120 vector can be digested, pcr purified and used directly for ligations. Treating with CIP or gel purifying the vector is unnecessary, because transformation of any intact V0120 plasmid into normal TOP10 competent cells (purple lid) will kill the host. V0120 can be successfully propagated in TOP10 Survival competent cells (green lid).
- V0120 is part# 116 in our internal database. Its annotated sequence can be found here.
- The annonated sequence of V0002 can be found here.
Sikorski Vectors: pRS304*, pRS305, pRS306
- We commonly use three Sikorski vectors to integrate constructs into the genomic yeast: pRS304* (TRP1), pRS305 (LEU2), and pRS306 (URA3). The 580a strain (SBy001, PSy580) contains the trp1[math]d[/math]63,leu2[math]d[/math]1, and ura3-52 mutant alleles, so successful integration at these loci confers the appropriate prototrophy. You can find more information about these alleles on the SGD.
- The pRS305 and pRS306 vectors are as described in R. S. Sikorski and P. Hieter, Genetics, 122: 19-27 (1989). The pRS304* vector is a modified version of the pRS304 vector (same reference).
- Currently, these vectors do not contain BioBrick ends, although they are compatible with Biobrick parts. See Strategy for Making Parts for more details.