Difference between revisions of "Part:BBa K880003"

(→‎A Utility for Assaying fim-Based Recombinases)
 
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=='''An Asymmetrically Digestible Reporter Utility for Assaying fim-Based Recombinases'''==
 
=='''An Asymmetrically Digestible Reporter Utility for Assaying fim-Based Recombinases'''==
  
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-IRL K137010 + Sequence with AgeI cut-site K165006 + IRR K137008 Mi8
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IRL K137010 + Sequence with AgeI cut-site K165006 + IRR K137008 Mi8
  
 
This is an asymmetrically digestible reporter, same orientation of inverted repeats as K137058 (external sites located internally of flip region)(IRL - “flip region” - IRR) for assaying fim-Based recombinase activity.
 
This is an asymmetrically digestible reporter, same orientation of inverted repeats as K137058 (external sites located internally of flip region)(IRL - “flip region” - IRR) for assaying fim-Based recombinase activity.
  
 
<p>https://static.igem.org/mediawiki/2012/e/e6/Asymmetrical_Digest_Reporter_Orientations.png</p>
 
<p>https://static.igem.org/mediawiki/2012/e/e6/Asymmetrical_Digest_Reporter_Orientations.png</p>
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<h2>Use of this Part in Characterizing the FimE Recombinase</h2>
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Team Michigan '12 used this part to characterize its recombinase-based switch systems.
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This asymmetrical digest reporter (K880003) in the vector pSB3C5 based on the inversion region found in K137058 was co-transformed in 10-beta <i>E. coli</i> (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2.  Colonies as well as broth subcultures of co-transformants were analyzed for inversion using the asymmetrical digest assay.  The asymmetrical digest reporter and <i>fimE</i> are present within the co-transformants; however, the asymmetrical digest reporter remains in the uninverted state (Fig 1, lane 3). 
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[[Image:MichFig5.png|350px|thumb|center|Fig 1.]]
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Scrutiny of the DNA sequence provided a possible explanation of the negative results.
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As constructed, verified by sequencing, the orientations of IRL(K137010) and IRR (K137008) within the inversion region of K137058 were orientated 180º when compared to the natural <i>fimS</i> region (Gally et al., 1996) and a previously engineered recombinase systems (Ham et al., 2006).  This resulted in the IRL and IRR being oriented such that the external half-sites are localized adjacent to the inversion region, while the internal half-sites are located externally (Fig 2).  We hypothesize that this 180º orientation of the IRL and IRR is the determinative factor to the inversion negative results.
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[[Image:MichFig6.png|350px|thumb|center|Fig 2.]]
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K880003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K880003 SequenceAndFeatures</partinfo>

Latest revision as of 01:38, 4 October 2012

An Asymmetrically Digestible Reporter Utility for Assaying fim-Based Recombinases

IRL K137010 + Sequence with AgeI cut-site K165006 + IRR K137008 Mi8

This is an asymmetrically digestible reporter, same orientation of inverted repeats as K137058 (external sites located internally of flip region)(IRL - “flip region” - IRR) for assaying fim-Based recombinase activity.

Asymmetrical_Digest_Reporter_Orientations.png

Use of this Part in Characterizing the FimE Recombinase

Team Michigan '12 used this part to characterize its recombinase-based switch systems.

This asymmetrical digest reporter (K880003) in the vector pSB3C5 based on the inversion region found in K137058 was co-transformed in 10-beta E. coli (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2. Colonies as well as broth subcultures of co-transformants were analyzed for inversion using the asymmetrical digest assay. The asymmetrical digest reporter and fimE are present within the co-transformants; however, the asymmetrical digest reporter remains in the uninverted state (Fig 1, lane 3).

Fig 1.

Scrutiny of the DNA sequence provided a possible explanation of the negative results. As constructed, verified by sequencing, the orientations of IRL(K137010) and IRR (K137008) within the inversion region of K137058 were orientated 180º when compared to the natural fimS region (Gally et al., 1996) and a previously engineered recombinase systems (Ham et al., 2006). This resulted in the IRL and IRR being oriented such that the external half-sites are localized adjacent to the inversion region, while the internal half-sites are located externally (Fig 2). We hypothesize that this 180º orientation of the IRL and IRR is the determinative factor to the inversion negative results.

Fig 2.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 116
  • 1000
    COMPATIBLE WITH RFC[1000]