Difference between revisions of "Part:BBa K2243024"

 
 
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<partinfo>BBa_K2243024 short</partinfo>
 
<partinfo>BBa_K2243024 short</partinfo>
  
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test the influence of attBP sites of Bxb1 to terminator 435
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To test the influence of attBP sites of Bxb1 to terminator ECK120034435 (abbreviation: 435) in the reverse direction.
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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 ECK120034435 (abbreviation: 435)  in the reverse 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 blocked.
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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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1. 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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2. We then characterized the inversion efficiency of Bxb1.
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We transformed the testing system containing terminator ECK120034435 (abbreviation: 435) in the reverse direction and expression system of Bxb1 recombinase into one single cell to see if the inversions happened, and if the leakage expression of Bxb1 would impact the system.
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Microplate spectrophotometer was used to conduct preliminary measurements. Bacterial culture was added into 96-well plate(200ul for each well). OD600 and fluorescence intensity were measured. Background OD600 and fluorescence of plate, culture medium ,and autofluorescence should be eliminated through setting control groups. Fluorescence intensity/OD600 was cauculated using the net fluorescence intensity and net OD600. The transcription barrier strength of attBP/Terminator can be characterized quantitatively referring to the Ts formula.
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[[File:Peking_1pair.png|600px|thumb|center|Fig.5 Terminators flanked by one pair of attB/P sites Induced with 0.1M IPTG and 10mM Arabinose]]
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The inversion efficiency was calculated according to the formula below.
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[[File:Peking_ie.png|600px|center]]
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<h2>Terminator Reference Table</h2>
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[[Media:Peking_trt.xlsx]] 
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2243024 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2243023 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2243024 parameters</partinfo>
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<partinfo>BBa_K2243023 parameters</partinfo>
 
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Latest revision as of 15:42, 1 November 2017


Bxb1 attB_435R_Bxb1 attP

To test the influence of attBP sites of Bxb1 to terminator ECK120034435 (abbreviation: 435) in the reverse direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of the terminator ECK120034435 (abbreviation: 435) in the reverse 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 blocked.

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

1. 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

2. We then characterized the inversion efficiency of Bxb1.

We transformed the testing system containing terminator ECK120034435 (abbreviation: 435) in the reverse direction and expression system of Bxb1 recombinase into one single cell to see if the inversions happened, and if the leakage expression of Bxb1 would impact the system.

Microplate spectrophotometer was used to conduct preliminary measurements. Bacterial culture was added into 96-well plate(200ul for each well). OD600 and fluorescence intensity were measured. Background OD600 and fluorescence of plate, culture medium ,and autofluorescence should be eliminated through setting control groups. Fluorescence intensity/OD600 was cauculated using the net fluorescence intensity and net OD600. The transcription barrier strength of attBP/Terminator can be characterized quantitatively referring to the Ts formula.

Fig.5 Terminators flanked by one pair of attB/P sites Induced with 0.1M IPTG and 10mM Arabinose

The inversion efficiency was calculated according to the formula below.

Peking ie.png

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.rc site found at 155