From CSBLwiki
Vectors
- pKD46
- Temperature sensitive replication (repA101ts) & conditional replicon (oriR101, requires Pir in trans for plasmid replciation)
- encodes lambda Red genes (exo, bet, gam)
- native terminator (tL3) after exo gene; arabinose-inducible promoter for expression (ParaB)
- encodes araC for repression of ParaB promoter
- Ampicillin resistant.
- oriR101 - pSC101 (repA-ts - a kind of plasmid resolvase; curable at 37~43C)
- While constitutive expression of the red gamma gene has a toxic effect in DH10B (recA-) cells under some conditions, thus limiting the efficiency of recombination, tightly regulated expression of the gamma gene together with simultaneous expression of the red alpha and beta genes allows efficient homologous recombination between linear DNA fragments and BACs resident in such cells as DH10B [1]
- pECM1
- pKT230
Replicons
(from Backbone plasmids nomenclature)
BioBrick plasmid backbone names take the form pSB#X#. The first number indicates the identity of the origin of replication. The number, corresponding replication origin, expected plasmid copy number and typical purpose of that origin are listed. To expand this list to include additional replication origins, obtain an account with the Registry of Standard Biological Parts and edit this page.
Origin of replication
| Number
| Replication origin
| Copy number
| Purpose
|
| 1
| modified pMB1 derived from pUC19
| 500-700
| Easy plasmid DNA purification
|
| 2
| F and P1 lytic derived from pSCANS-1-BNL
| 1-2 inducible to high copy
| Inducible copy number
|
| 3
| p15A derived from pMR101
| 10-12
| Multi-plasmid engineered systems
|
| 4
| rep101, repA derived from pSC101
| ~5
| Small cell to cell copy number variation
|
| 5
| derived from F plasmid
| 1-2
| Improved plasmid stability
|
| 6
| pMB1 derived from pBR322
| 15-20
| Multi-plasmid engineered systems
|
Links
- Addgene Plasmid Tools
- NEB plasmid Table (maps & sites)
- Novagen pET vector Table
- pBAD manual
- E. coli Host For cloning and transformation, we recommend using a recA, endA strain such as TOP10 (included in the kit, also available separately; see page vii). This strain is capable of transporting L-arabinose, but not metabolizing it. This is important for expression studies as the level of L-arabinose will be constant inside the cell and not decrease over time. Please note that other strains may be suitable for general use. Be sure to check the genotype of your strain. It should be araBADC- and araEFGH+ (Bachmann, 1990). The E. coli strain LMG194 is included in the kit to ensure low basal level expression of toxic genes (Guzman et al., 1995). This strain is capable of growth on minimal medium (RM medium), which allows additional repression of PBAD by glucose. Once you have determined that you have the correct construct, transform it into LMG194 prior to performing expression experiments.
- Genotype of TOP10: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araA-leu)7697 galU galK rpsL endA1 nupG. Note: This strain is araBADC–. It is deleted for both araBA and araC, and the gene for araD has a point mutation in it, making it inactive.
- Genotype of LMG194: F- ΔlacX74 gal E thi rpsL ΔphoA (Pvu II) Δara714 leu::Tn10. Please note that this strain is streptomycin and tetracycline resistant.
