Difference between revisions of "Part:BBa K1433011"
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<partinfo>BBa_K1433011 short</partinfo>
 
<partinfo>BBa_K1433011 short</partinfo>
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<p>This part is a circuit designed to test the function of Bxb1 gp35. Bxb1 gp35 is a serine integrase of Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP and the formation of new sites attL and attR.</p>
  
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    This part is a circuit which designed to test function of Bxb1 gp35. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase along can typically catalyzes site-specific recombination between attB and attP, the attachment sites on the phage chromosome and host chromosome. This recombination result in reverse of the sequence between attB and attP, then form new sites attL and attR.  
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https://static.igem.org/mediawiki/parts/e/ee/ZJU_int-xis-bplr.gif
  
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This part is composed of 7 elements.
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<p><b><big>Composition:</big></b><br />
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    1.Terminator(reverse): BBa_B0015, a strong double terminator.
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<ol><li>Terminator (reverse): [https://parts.igem.org/Part:BBa_B0015 BBa_B0015], a strong double terminator.</li>
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    2.RFP(reverse): J06504, a monomeric RFP optimized for bacteria.
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<li>RFP (reverse): [https://parts.igem.org/Part:BBa_J06504 BBa_J06504], a monomeric RFP optimized for bacteria.</li>
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    3.RBS: BBa_B0034, a strong RBS.
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<li>RBS: [https://parts.igem.org/Part: BBa_B0034 BBa_B0034], a strong RBS.</li>
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    4.attB and attP sites: Recognition site for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.
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<li>attB and attP sites: Recognition sites for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.</li>
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    5.Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
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<li>Promoter: [https://parts.igem.org/Part:BBa_J23110 BBa_J23110], a middle-ground Bacterial constitutive promoter.</li>
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    6.GFP: BBa_E0040, green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.
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<li>GFP:  [https://parts.igem.org/Part:BBa_E0040 BBa_E0040], green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.</li>
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    7.Terminator: BBa_B0015, a strong double terminator.
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<li>Terminator: [https://parts.igem.org/Part:BBa_B0015 BBa_B0015], a strong double terminator.</li>
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</ol></p>
  
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    This circuit can not express Bxb1 gp35 nor work along. When this part work with BBa_K1433014 or BBa_K1433015, which can express Bxb1 gp35. Bxb1 serine integrase gp35 will mediate reverse of the promoter between attB and attP sites, then change it to new attL and attR sites.
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[[File:ZJU set overview.jpg]]
  
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    There are two reporter gene on this circuit, GFP and RFP. At first, the promoter between attB and attP sites is forward and can promote expression of downstream GFP gene to make bacteria looks green. When co-transform of this part and BBa_K1433014 or BBa_K1433015, gp35 will express to reverse the promoter. The reversed promoter can promote the expression of upstream RFP gene, the color change of bacterial colony or solution can be easily observed.
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<p>This circuit can neither express Bxb1 gp35 nor work along. When this part works with [https://parts.igem.org/Part:BBa_K1433014 BBa_K1433014] or [https://parts.igem.org/Part:BBa_K1433015 BBa_K1433015], Bxb1 gp35 can be expressed, which will then mediate the reverse of the promoter between attB and attP, and change it to new attL and attR.</P>
  
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    Co-transformation of this part and BBa_K1433018 can be used to test the background expression of gp35. BBa_K1433018 contains two BBa_B0015 terminator between two homologous arms to suppress the expression of gp35, The co-transformed bacteria should be pure green. Indeed, there are little bacteria present red or mix of green and red because of background leakage. Lambda red is a Lambda phage derived recombination system, it can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA are flanked by 36-50bp homologous arms. Lambda red mediate  homologous recombination can replace two terminators with other sequence or gene to insert exogenous genes and gp35 depression make the color change of green to red. Measuring this leakage of gp35 is significant in recombination experiment cause the real reverse ability of gp35 should get rid of interference of background leakage.
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<p><b><big>INT+SET</big></b><br />
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There are two reporter genes on the circuit, GFP and RFP. At first, the promoter between attB and attP sites is in forward direction and can promote the expression of downstream GFP gene to make bacteria green. When the co-transform of this part and [https://parts.igem.org/Part:BBa_K1433014 BBa_K1433014] or [https://parts.igem.org/Part:BBa_K1433015 BBa_K1433015] occurs, gp35 will express and then reverse the promoter, which will promote the expression of upstream RFP gene. The change of color of bacterial colony or solution can be easily observed.</p>
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<p><b><big>INT+SOCKET</big></b><br />
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Co-transformation of this part and [https://parts.igem.org/Part:BBa_K1433018 BBa_K1433018] can be used to test the background expression of gp35. [https://parts.igem.org/Part:BBa_K1433018 BBa_K1433018] contains two [https://parts.igem.org/Part:BBa_B0015 BBa_B0015] terminators between two homologous arms, which can suppress the expression of gp35. The co-transformed bacteria should be pure green. Indeed, there are a few bacteria presenting red or mix of green and red because of background leakage. Lambda red is a Lambda-phage-derived recombination system, which can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. Lambda-red-mediated homologous recombination can replace two terminators with exogenous sequences, thus removing gp35 depression and changing the color from green to red. Measuring this leakage of gp35 is important in recombination experiment because the real reverse ability of gp35 should be under no interference of background leakage.</p>
  
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 12:38, 17 October 2014

Terminator-RFP-RBS-attB-P-attP-RBS-GFP-Terminator

This part is a circuit designed to test the function of Bxb1 gp35. Bxb1 gp35 is a serine integrase of Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP and the formation of new sites attL and attR.

ZJU_int-xis-bplr.gif

Composition:

  1. Terminator (reverse): BBa_B0015, a strong double terminator.
  2. RFP (reverse): BBa_J06504, a monomeric RFP optimized for bacteria.
  3. RBS: BBa_B0034 BBa_B0034, a strong RBS.
  4. attB and attP sites: Recognition sites for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.
  5. Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
  6. GFP: BBa_E0040, green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.
  7. Terminator: BBa_B0015, a strong double terminator.

ZJU set overview.jpg

This circuit can neither express Bxb1 gp35 nor work along. When this part works with BBa_K1433014 or BBa_K1433015, Bxb1 gp35 can be expressed, which will then mediate the reverse of the promoter between attB and attP, and change it to new attL and attR.


INT+SET
There are two reporter genes on the circuit, GFP and RFP. At first, the promoter between attB and attP sites is in forward direction and can promote the expression of downstream GFP gene to make bacteria green. When the co-transform of this part and BBa_K1433014 or BBa_K1433015 occurs, gp35 will express and then reverse the promoter, which will promote the expression of upstream RFP gene. The change of color of bacterial colony or solution can be easily observed.


INT+SOCKET
Co-transformation of this part and BBa_K1433018 can be used to test the background expression of gp35. BBa_K1433018 contains two BBa_B0015 terminators between two homologous arms, which can suppress the expression of gp35. The co-transformed bacteria should be pure green. Indeed, there are a few bacteria presenting red or mix of green and red because of background leakage. Lambda red is a Lambda-phage-derived recombination system, which can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. Lambda-red-mediated homologous recombination can replace two terminators with exogenous sequences, thus removing gp35 depression and changing the color from green to red. Measuring this leakage of gp35 is important in recombination experiment because the real reverse ability of gp35 should be under no interference of background leakage.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 933
    Illegal NheI site found at 956
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 880
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 900
    Illegal BsaI.rc site found at 983
    Illegal BsaI.rc site found at 1684