Difference between revisions of "Part:BBa J61001:Experience"
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In particular, <partinfo>BBa_K300008</partinfo> 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). | In particular, <partinfo>BBa_K300008</partinfo> 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 (<partinfo>BBa_K300984</partinfo>) and BW23474 (<partinfo>BBa_K300985</partinfo>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<partinfo>BBa_V1001</partinfo>) and MC1061 (<partinfo> | + | BW25141 (<partinfo>BBa_K300984</partinfo>) and BW23474 (<partinfo>BBa_K300985</partinfo>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<partinfo>BBa_V1001</partinfo>) and MC1061 (<partinfo>BBa_K300078</partinfo>) were chosen as pir- strains. |
Revision as of 17:23, 19 October 2010
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Applications of BBa_J61001
User Reviews
UNIQa2af388f7eb35507-partinfo-00000000-QINU
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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) and MC1061 (BBa_K300078) were chosen as pir- strains.
and plated on LB+Cm at 34 ug/ml.
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+ or 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. |
UNIQa2af388f7eb35507-partinfo-00000011-QINU