Difference between revisions of "Part:BBa K2243035"
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===Usage and Biology===
 
===Usage and Biology===
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Usage
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We constructed this part to characterize the recombination efficiency of the recombinase Lactococcal phage TP901-1. It consists of the terminator ECK120034435 (abbreviation: 435) in the reverse direction flanked by attB and attP sites of recombinase TP901-1. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated.
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Biology
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The attP site of TP901-1 is used to integrate phage DNA at the host attB site of Lactococcal 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.
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Characterization
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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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And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.
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[[File:Peking IPTG 0.1M.png|200px|thumb|center|Terminator Strength Induced with 0.1M IPTG]]
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[[File:Peking IPTG 1M.png|200px|thumb|center|Terminator Strength Induced with 1M IPTG]]
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2. We then characterized the inversion efficiency.
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We transformed the testing system and expression system of corresponding recombinase into one single cell to see if the inversions happened, and if the leakage expression of recombinases would impact the system. In the end, couples of terminators and recombinases with near complete inversions and minimal leakage were selected.
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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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Revision as of 16:11, 29 October 2017


TP901-1 attB_435R_TP901-1 attP

To test the influence of attBP sites of TP901-1 to terminator ECK120034435 (abbreviation: 435) in the reverse direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Lactococcal phage TP901-1. It consists of the terminator ECK120034435 (abbreviation: 435) in the reverse direction flanked by attB and attP sites of recombinase TP901-1. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated.

Biology

The attP site of TP901-1 is used to integrate phage DNA at the host attB site of Lactococcal 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


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

Terminator Strength Induced with 0.1M IPTG
Terminator Strength Induced with 1M IPTG

2. We then characterized the inversion efficiency.

We transformed the testing system and expression system of corresponding recombinase into one single cell to see if the inversions happened, and if the leakage expression of recombinases would impact the system. In the end, couples of terminators and recombinases with near complete inversions and minimal leakage were selected.

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.





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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]