Difference between revisions of "Part:BBa K2243031"
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===Characterization===
 
===Characterization===
  
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We first characterized the terminator strength using the following formula:
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1. We first characterized the terminator strength using the following formula:
  
  
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[[File:Peking TS good terminators.png|600px|thumb|center|Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG]]
 
[[File:Peking TS good terminators.png|600px|thumb|center|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 phiC31.
  
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We transformed the testing system containing terminator ECK120034435 (abbreviation: 435) in the forward direction and expression system of phiC31 recombinase into one single cell to see if the inversions happened, and if the leakage expression of phiC31 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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Terminator Reference Table
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[[Media:Peking_trt.xlsx]]  
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[[File:Peking_1pair.png|600px|thumb|center|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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Terminator Reference Table
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[[Media:Peking_trt.xlsx]]
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2243031 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2243023 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2243031 parameters</partinfo>
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<partinfo>BBa_K2243023 parameters</partinfo>
 
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Revision as of 14:59, 1 November 2017


phiC31 attB_435F_phiC31 attP

To test the influence of attB/P sites of phiC311 to terminator ECK120034435 (abbreviation: 435) in the forward direction.


Usage and Biology

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Streptomyces bacteria phage phiC31. It consists of the terminator ECK120034435 (abbreviation: 435) in the forward direction flanked by attB and attP sites of recombinase phiC31. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated.

Biology

The attP site of phiC31 is used to integrate phage DNA at the host attB site of Streptomyces bacterium 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

Terminator Strength Induced with 0.1mM IPTG
Terminator Strength Induced with 1mM IPTG

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

Terminator Strength Induced with 0.1M IPTG
Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG

2. We then characterized the inversion efficiency of phiC31.

We transformed the testing system containing terminator ECK120034435 (abbreviation: 435) in the forward direction and expression system of phiC31 recombinase into one single cell to see if the inversions happened, and if the leakage expression of phiC31 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.

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