Difference between revisions of "Part:BBa K3254005"

 
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===Usage and Biology===
 
===Usage and Biology===
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==Visual Result as a Normally Closed Switch==
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*We conducted a simple test to see if our design met the expection.
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===Experimental Setup===
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*Genetic design principle of the experimental group is described on the page of [[Part:BBa_K3254013|BBa_K3254013]].
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*A P15A-AmpR plasmid was co-transfered into the E.coli DH5α host cell with the reporter plasmid containing this part as the negative control.
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*Single colonies were selected from the experimental LB-agar plate and negative control LB-agar plate, then inoculated into EP tubes with 500 μL M9 supplemented medium containing 500 μM IPTG for overnight growth at 37 °C and 200 rpm.
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*Tubes were centrifuged at 10000g for 1 min. Then observed the GFP fluorescence of the cell precipitations under blue light.
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===Results===
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*IBR-C35/F55/S37/E21/T25/G22 indicate the experimental systems for phiC31/Int5/Int7/Int8/Int10/TG1 respctively.
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*We observed the GFP fluorescence from the experimental tube as expected.<br>
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[[File:T--GENAS_China--primary_screening.png|600px|thumb|center| ]]<br>
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==Quantitative Characterizaion of the Normally Open Switch==
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===Experimental Setup===
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*Bacteria harboring the circuits (see the top part of the result image) first inoculated from single colonies into a flat-bottom 96-well plate for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with 500 μM IPTG inducer and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, 3-μL samples each culture were transferred to a new 96-well plate containing 200 μL per well of PBS supplemented with 2 mg/mL kanamycin.
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*The fluorescence distribution of each sample was assayed using a flow cytometry. The arithmetical mean of each sample was determined using FlowJo software.
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*The principle of data processing is shown on the result image.
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===Results===
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*IBR-C35/F55/S37/E21/T25/G22 indicate the experimental systems for phiC31/Int5/Int7/Int8/Int10/TG1 respctively.
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*Compared to other parts, this part performed well.<br>
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[[File:T--GENAS_China--primary_quantitative_characterizaion.png|600px|thumb|center| ]]<br>
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==Orthogonality Characterization==
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===Genetic Design===
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*The composition and principle of the experimental system are indicated below.
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[[File:T--GENAS_China--excision_with_backbone.PNG|200px|thumb|left| ]]
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===Experimental Setup===
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*The reporter plasmid contained this part were co-transferred into E.coli DH5α host with 6 integrase generator plasmids.
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Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with 500 μM IPTG inducer and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, The cultures were sampled for genotype PCR testing.
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*The principle of genotype identification was shown on the right of results image.
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===Results===
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*IBR-G22 was the plasmid containing this part.
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*The result indicates that this part can only be recombined by TG1 integrase.
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*The sequence (attL or attR) after recombination is GATCAGCTCCGCGGGCAAGACCGTGCTCTTACCCAGTTGGGCGGGA.
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[[File:T--GENAS_China--orthogonality_test.png]]
  
 
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Revision as of 12:15, 20 October 2019


TG1attB-BsaI site-terminator-TG1attP

We expected that this part can be placed between a promoter and a translational unit part and work as a normally open (NO) switch for the downstreamed gene. and switch to ON state by excising the terminator L3S3P22 between the att sites when it reacted with TG1 integrase (BBa_K2460007). Two BsaI restriction site were added between attB site and terminator. As a result, it can work as a normally closed (NC) switch for the gene which was inserted between the two BsaI site and switch to OFF state when it was excised.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 60
    Illegal BsaI.rc site found at 48