Difference between revisions of "Part:BBa K1742009"

 
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<partinfo>BBa_K1742009 short</partinfo>
 
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Composite composed of the recombinase Bxb1 optimize for Nicothiana Benthamiana and a reporter to prove its action. The reporter composite consist in a terminator flankeated by the recognition sequence of Bxb1 between the promoter and the CDS. Only if the recombinase is present, the GFP will be observed.
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Bxb1 is a protein from ''Mycobateriophage'' Bxb1’s gp35 gen. Its function is to regulate the lysogenic cycle of the phage by integrating and excising phage’s genome in ''Mycobacterium smegmatis'' chromosome [1]. This integrase is able to recognize two different sites, one in phage’s genome (attP, “Phage attachment site”) and another in bacterial chromosome (attB, “Bacterial attachment site”)[2]. Depending on the position and sense of these sites, bxb1 is able of excising or inverting. We used bxb1 as excisionase by flanking a sequence close to the promoter
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== Biology and Usage ==
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The plant codon-optimized BxBI gene ([https://parts.igem.org/Part:BBa_K1742005 BBa_K1742005]) was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector.  The BxbI integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S).
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In order to see the activity of the integrase, Valencia_UPV 2015 designed a reporter element ([https://parts.igem.org/Part:BBa_K1742006 BBa_K1742006]) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attB:T35S:attP:omegaUTR) of BxbI. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator.  
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With a binary GoldenBraid assembly step we obtained the present multigenic construct ([https://parts.igem.org/Part:BBa_K1742009 BBa_K1742009]) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into ''N. benthamiana'' plants for testing the expression of GFP.  
  
 
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Revision as of 02:03, 17 September 2015

35S:BxbI:T35S - 35S:ReporterBxBI::GFP:T35S

Bxb1 is a protein from Mycobateriophage Bxb1’s gp35 gen. Its function is to regulate the lysogenic cycle of the phage by integrating and excising phage’s genome in Mycobacterium smegmatis chromosome [1]. This integrase is able to recognize two different sites, one in phage’s genome (attP, “Phage attachment site”) and another in bacterial chromosome (attB, “Bacterial attachment site”)[2]. Depending on the position and sense of these sites, bxb1 is able of excising or inverting. We used bxb1 as excisionase by flanking a sequence close to the promoter

Biology and Usage

The plant codon-optimized BxBI gene (BBa_K1742005) was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector. The BxbI integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S).

In order to see the activity of the integrase, Valencia_UPV 2015 designed a reporter element (BBa_K1742006) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attB:T35S:attP:omegaUTR) of BxbI. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator. With a binary GoldenBraid assembly step we obtained the present multigenic construct (BBa_K1742009) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into N. benthamiana plants for testing the expression of GFP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1959
    Illegal BamHI site found at 1141
    Illegal XhoI site found at 660
    Illegal XhoI site found at 1146
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
    Illegal NgoMIV site found at 49
    Illegal NgoMIV site found at 1460
    Illegal NgoMIV site found at 1698
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2170