Difference between revisions of "Part:BBa K2243018"
 
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<partinfo>BBa_K2243018 short</partinfo>
 
<partinfo>BBa_K2243018 short</partinfo>
  
 
To test the influence of Bxb1 attB/P to terminator L3S3P22 (abbreviation: 322) in the forward direction.
 
To test the influence of Bxb1 attB/P to terminator L3S3P22 (abbreviation: 322) in the forward direction.
  
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===Usage and Biology===
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<h2>Usage</h2>
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We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of the terminator L3S3P22 (abbreviation: 322)  in the forward direction flanked by attB and attP sites of recombinase Bxb1 gp35. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated, and upstream sequence is transcribed.
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<h2>Biology</h2>
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The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the terminator, attB and attP sites by oligo synthesis.
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<h2>Characterization</h2>
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We first characterized the terminator strength using the following formula:
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Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator
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[[File:Peking_TS_0.1mM.png|800px|thumb|center|Fig.1 Terminator Strength Induced with 0.1mM IPTG]]
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[[File:Peking_TS_1mM.png|800px|thumb|center|Fig.2 Terminator Strength Induced with 1mM IPTG]]
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And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.
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[[File:Peking ratio.png|600px|thumb|center|Fig.3 Terminator Strength Induced with 0.1M IPTG]]
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[[File:Peking TS good terminators.png|600px|thumb|center|Fig.4 Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG]]
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<h2>Terminator Reference Table</h2>
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[[Media:Peking_trt.xlsx]]
  
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We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of a constitutive promoter (BBa_J23119) flanked by attB and attP sites of recombinase Bxb1 gp35. Different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. Upon recombination, the orientation of the constitutive promoter change. As a result, expression of downstream sequence is shut down, and upstream sequence is transcribed.
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2243018 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2243018 SequenceAndFeatures</partinfo>
  

Latest revision as of 15:43, 1 November 2017


Bxb1 attB_322F_Bxb1 attP

To test the influence of Bxb1 attB/P to terminator L3S3P22 (abbreviation: 322) in the forward direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of the terminator L3S3P22 (abbreviation: 322) in the forward direction flanked by attB and attP sites of recombinase Bxb1 gp35. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated, and upstream sequence is transcribed.

Biology

The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the terminator, attB and attP sites by oligo synthesis.

Characterization

We first characterized the terminator strength using the following formula:


Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator

Fig.1 Terminator Strength Induced with 0.1mM IPTG
Fig.2 Terminator Strength Induced with 1mM IPTG

And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.

Fig.3 Terminator Strength Induced with 0.1M IPTG
Fig.4 Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG

Terminator Reference Table

Media:Peking_trt.xlsx


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 NgoMIV site found at 4
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 24
    Illegal BsaI site found at 103
    Illegal BsaI.rc site found at 147