Difference between revisions of "Part:BBa J61001:Experience"
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*self-ligated <partinfo>BBa_K300008</partinfo>. | *self-ligated <partinfo>BBa_K300008</partinfo>. | ||
− | and plated on LB+Cm at 34 ug/ml. | + | and plated on LB+Cm at 34 ug/ml for high-copy plasmids and Cm at 12.5 ug/ml for medium/low copy plasmids. |
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− | These results show that <partinfo>BBa_J61001</partinfo> replication origin can be only propagated in pir+ | + | These results show that <partinfo>BBa_J61001</partinfo> replication origin can be only propagated in pir+ and pir-116 strains (<partinfo>BBa_K300084</partinfo> and <partinfo>BBa_K300085</partinfo>), 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. | 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. |
Revision as of 08:33, 22 October 2010
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Applications of BBa_J61001
User Reviews
UNIQ18f7104f88ef22cd-partinfo-00000000-QINU
••••
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) and MC1061 (BBa_K300078) were chosen as pir- strains.
and plated on LB+Cm at 34 ug/ml for high-copy plasmids and Cm at 12.5 ug/ml for medium/low copy plasmids.
efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng] The results are shown here:
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. |
UNIQ18f7104f88ef22cd-partinfo-00000011-QINU