Difference between revisions of "Part:BBa K3254007"
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This is a serine-type phage (LSTP) integrases. The original organism is the Geobacillus sp. Y412MC61. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part [[Part:BBa_K3254002|BBa_K3254002]]).  
 
This is a serine-type phage (LSTP) integrases. The original organism is the Geobacillus sp. Y412MC61. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part [[Part:BBa_K3254002|BBa_K3254002]]).  
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=Orthogonality Characterization=
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We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases include phiC31, Int5, Int8, Int10 and TG1.
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==Genetic design==
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The composition and principle of the experimental system are indicated below. More details can be seen on the pages of [[Part:BBa_K3254027|BBa_K3254027]] and [[Part:BBa_K3254002|BBa_K3254002]].
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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 plasmid containing the Int5 expressional unit was co-transfered into E.coli DH5α host with 6 reporter plasmids containing different attP-attB sites sequences.
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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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The result indicate that Int7 integrase has a good orthogonality with the other 5 att sites and compatible with other integrases. IBR-C35/F55/S37/E21/T25/G22 were the plasmids with phiC31/Int5/Int7/Int8/Int10/TG1 att sites.
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[[File:T--GENAS_China--orthogonality_test.png]]
  
 
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Revision as of 12:49, 19 October 2019


Integrase Int7

This is a serine-type phage (LSTP) integrases. The original organism is the Geobacillus sp. Y412MC61. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part BBa_K3254002).

Orthogonality Characterization

We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases include phiC31, Int5, Int8, Int10 and TG1.

Genetic design

The composition and principle of the experimental system are indicated below. More details can be seen on the pages of BBa_K3254027 and BBa_K3254002.

T--GENAS China--excision with backbone.PNG

Experimental Setup

The plasmid containing the Int5 expressional unit was co-transfered into E.coli DH5α host with 6 reporter plasmids containing different attP-attB sites sequences. 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. The principle of genotype identification was shown on the right of results image.

Results

The result indicate that Int7 integrase has a good orthogonality with the other 5 att sites and compatible with other integrases. IBR-C35/F55/S37/E21/T25/G22 were the plasmids with phiC31/Int5/Int7/Int8/Int10/TG1 att sites.

T--GENAS China--orthogonality test.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 153
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 814
    Illegal AgeI site found at 1215
  • 1000
    COMPATIBLE WITH RFC[1000]