Designed by: John Anderson   Group: Arkin Lab   (2006-07-31)

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Applications of BBa_J61001

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UNIPV-Pavia iGEM 2010

BBa_K300008 was used as a validation construct for this conditional replication origin, in order to test its capability to be propagated in pir+ or pir-116 strain and its inability to propagate in the other E. coli strains.

In particular, BBa_K300008 was cut with XbaI-SpeI and the insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid).

BW25141 (BBa_K300984) and BW23474 (BBa_K300985) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (BBa_V1001), MC1061 (BBa_K300078) and MG1655 (BBa_V1000) were chosen as pir- strains.

All these strains were made competent following the commonly used CaCl2 method [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Then, a vial of 100 ul of competent cells was transformed with 2-4 ng of:

  • no DNA (negative control);
  • a pSB*** series vector (positive control);
  • self-ligated BBa_K300008.

and plated on LB+Cm at 34 ug/ml for high-copy plasmids, Cm at 12.5 ug/ml for medium/low copy plasmids and for the negative control strains transformed with the R6K plasmids.

The colonies were counted in each plate and the transformation efficiency was estimated in [CFU/ug of DNA] as:

efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]

The results are shown here:

Strain Efficiency with no DNA Efficiency with pSB*** (positive control) Efficiency with the self-ligated BBa_K300008 (R6K plasmid)
BBa_K300084 0 10^5 10^5
BBa_K300085 0 10^6 10^6
BBa_V1001 0 10^8 0
BBa_K300078 0 10^6 0
BBa_V1000 0 10^5 0

These results show that BBa_J61001 replication origin can be only propagated in pir+ and pir-116 strains (BBa_K300084 and BBa_K300085), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.

Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.