Difference between revisions of "Part:BBa K1077005"
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The fim transcriptor is capable of changing states completely and unidirectionally
 
The fim transcriptor is capable of changing states completely and unidirectionally
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fig. 2
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NEB 10-beta E. coli, which lack the native fim switch (fimS), were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). E. coli expressing hbiF recombnase constitutively displayed a deep blue phenotype (A). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation. E. coli expressing fimE recombinase constitutively displayed a deep blue phenotype (fig. x). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation.
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The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry, showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE).
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Fig. 1 - The fim transcriptor is capable of changing states completely and unidirectionally
  
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NEB 10-beta E. coli, which lack the native fim switch (fimS) and known fim recombinases, were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry in A and B showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation and fime’s previously observed functionality of catalyzing the inversion of fimS from the ON to OFF orientation. C and D show the digest assay fragments. The faint band in D, seemingly indicating residual OFF state, may correspond to the constitutive recombinase generator which is about the same size. We did not have time to cure the bacteria of the plasmid. Additionally, the switch is on the high copy pSB1C3 plasmid and so it could be that some switch plasmids are escaping recombination. We did not have time to move the switch to a low copy plasmid or the chromosome.
  
 
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Revision as of 22:39, 28 September 2013

J23100 fim switch ON orientation

The fim transcriptor is capable of changing states completely and unidirectionally Sdsadsafdsa.jpg

Fig. 1 - The fim transcriptor is capable of changing states completely and unidirectionally

NEB 10-beta E. coli, which lack the native fim switch (fimS) and known fim recombinases, were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation). The state of the switch was assayed by using an asymmetric digest assay. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. The digest assay was quantified using densitometry in A and B showing greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation and fime’s previously observed functionality of catalyzing the inversion of fimS from the ON to OFF orientation. C and D show the digest assay fragments. The faint band in D, seemingly indicating residual OFF state, may correspond to the constitutive recombinase generator which is about the same size. We did not have time to cure the bacteria of the plasmid. Additionally, the switch is on the high copy pSB1C3 plasmid and so it could be that some switch plasmids are escaping recombination. We did not have time to move the switch to a low copy plasmid or the chromosome.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 306
    Illegal NheI site found at 329
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]