Difference between revisions of "Part:BBa K2787011"
(→‎Usage and Biology)
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===Usage and Biology===
 
===Usage and Biology===
 +
a plasmid with GFP under control of A2 orthogonal RBS with B8 orthogonal 16s rRNA is constructed to test the orthogonality of C9 RBS.
 
[[File:Shanghaitech2018-oribo-orbs-07-07.png|700px|center]]
 
[[File:Shanghaitech2018-oribo-orbs-07-07.png|700px|center]]
 +
<br>
 
[[File:ShanghaiTech2018-c9_rbs.png|700px|center]]
 
[[File:ShanghaiTech2018-c9_rbs.png|700px|center]]
 +
FP, the target gene downstream the C9 orthogonal RBS is activated better by the A2 orthogonal ribosome even than the C9 ribosome. The expression of GFP increases slightly over time. This shows that the orthogonality of the C9 orthogonal RBS does not work as expected.<br>
 +
 
[[File:ShanghaiTech2018-A2 16S.png|700px|center]]
 
[[File:ShanghaiTech2018-A2 16S.png|700px|center]]
 +
The GFP under control of different orthogonal RBS is tested within A2 orthogonal ribosomes. The result shows that the target gene will not be activated by the A2 ribosome unless it is downstream the A2 RBS, which shows great orthogonality of A2 ribosome from others.
 +
 
[[File:A2-B8-C9-Orthogonality.png|700px|center]]
 
[[File:A2-B8-C9-Orthogonality.png|700px|center]]
 +
The figure shows the orthogonality overview between the different ribosomes and RBSs. It can be seen that the orthogonality of A2 ribosome is very high, which is well prepared for using.
 +
 +
 +
 +
===Usage and Biology===
 +
a plasmid with GFP under control of A2 orthogonal RBS with B8 orthogonal 16s rRNA is constructed to test the orthogonality of A2 RBS.
 +
[[File:Shanghaitech2018-oribo-orbs-07-07.png|500px|center]]
 +
<br>
 +
[[File:ShanghaiTech2018-b8 rbs.png|700px|center]]
 +
GFP, the target gene downstream the A2 orthogonal RBS is activated a little bit by the C9 orthogonal ribosome. The expression of GFP increases slighty over time. This shows that the orthogonality between the C9 orthogonal 16s rRNA and the B8 orthogonal RBS is worse than that between B8 orthogonal RBS and other orthogonal ribosomes<br>
 +
 +
[[File:ShanghaiTech2018-A2 16S.png|700px|center]]
 +
The figure shows great orthogonality between B8 orthogonal RBS and other orthogonal ribosomes. However, the orthogonality between the B8 RBS and the A2 ribosome is a little worse than others.
 +
 +
[[File:A2-B8-C9-Orthogonality.png|700px|center]]
 +
The figure shows the orthogonality overview between the different ribosomes and RBSs.
 +
 +
 +
 +
 +
===Usage and Biology===
 +
 +
[[File:ShanghaiTech2018-A2 16s.png|700px|center]]
 +
 +
[[File:ShanghaiTech2018-A2 A2.png|700px|center]]
 +
GFP, the target gene downstream the A2 orthogonal RBS is highly activated by the A2 orthogonal ribosome. The expression of GFP increases gradually over time. In the absence of A2 orthogonal ribosome, the GFP is repressed deeply, which shows the high orthogonality of A2 orthogonal RBS and A2 orthogonal ribosome.<br>
 +
 +
 +
 +
 +
[[File:ShanghaiTech2018-A2 rbs.png|700px|center]]
 +
The GFP under control of A2 orthogonal RBS is tested within different 16s rRNAs. The fluorescence with A2 orthogonal ribosome is the best among host ribosomes and A2,B8,C9 ribosomes, which shows the good orthogonality of A2 16s rRNA with A2 orthogonal RBS.
  
 +
[[File:ShanghaiTech2018-A2.png|700px|center]]
 +
[[File:A2-B8-C9-Orthogonality.png|500px|center]]
 +
The fluorescence under the test with 3 different orthogonal RBS and 3 different orthogonal ribosmes. It shows that the orthogonality of A2 ribosome and A2 RBS is one of the best ones.
 +
[[File:Shanghaitech-A2.png|450px|center<b>Fig. 3 <br>
 +
A2 O-16S fluorescence at 200 minutes, showing that the exist of A2 orthogonal 16s rRNA will greatly activate the GFP. However, it will be deeply depressed without the A2 ribosome.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 03:24, 18 October 2018


GFP under C9 orthogonal RBS with orthogonal 16S rRNA A2

The 16S rRNA A2 under constitutive promoter BBa_J23100(BBa_K2787006) and pCons-oRBSC9-GFP(BBa_K2787002) are linked into one plasmid to quantify the orthogonality of the A2 and C9 ribosome system.


Usage and Biology

a plasmid with GFP under control of A2 orthogonal RBS with B8 orthogonal 16s rRNA is constructed to test the orthogonality of C9 RBS.

Shanghaitech2018-oribo-orbs-07-07.png


ShanghaiTech2018-c9 rbs.png

FP, the target gene downstream the C9 orthogonal RBS is activated better by the A2 orthogonal ribosome even than the C9 ribosome. The expression of GFP increases slightly over time. This shows that the orthogonality of the C9 orthogonal RBS does not work as expected.

ShanghaiTech2018-A2 16S.png

The GFP under control of different orthogonal RBS is tested within A2 orthogonal ribosomes. The result shows that the target gene will not be activated by the A2 ribosome unless it is downstream the A2 RBS, which shows great orthogonality of A2 ribosome from others.

A2-B8-C9-Orthogonality.png

The figure shows the orthogonality overview between the different ribosomes and RBSs. It can be seen that the orthogonality of A2 ribosome is very high, which is well prepared for using.


Usage and Biology

a plasmid with GFP under control of A2 orthogonal RBS with B8 orthogonal 16s rRNA is constructed to test the orthogonality of A2 RBS.

Shanghaitech2018-oribo-orbs-07-07.png


ShanghaiTech2018-b8 rbs.png

GFP, the target gene downstream the A2 orthogonal RBS is activated a little bit by the C9 orthogonal ribosome. The expression of GFP increases slighty over time. This shows that the orthogonality between the C9 orthogonal 16s rRNA and the B8 orthogonal RBS is worse than that between B8 orthogonal RBS and other orthogonal ribosomes

ShanghaiTech2018-A2 16S.png

The figure shows great orthogonality between B8 orthogonal RBS and other orthogonal ribosomes. However, the orthogonality between the B8 RBS and the A2 ribosome is a little worse than others.

A2-B8-C9-Orthogonality.png

The figure shows the orthogonality overview between the different ribosomes and RBSs.



Usage and Biology

700px
ShanghaiTech2018-A2 A2.png

GFP, the target gene downstream the A2 orthogonal RBS is highly activated by the A2 orthogonal ribosome. The expression of GFP increases gradually over time. In the absence of A2 orthogonal ribosome, the GFP is repressed deeply, which shows the high orthogonality of A2 orthogonal RBS and A2 orthogonal ribosome.



ShanghaiTech2018-A2 rbs.png

The GFP under control of A2 orthogonal RBS is tested within different 16s rRNAs. The fluorescence with A2 orthogonal ribosome is the best among host ribosomes and A2,B8,C9 ribosomes, which shows the good orthogonality of A2 16s rRNA with A2 orthogonal RBS.

700px
A2-B8-C9-Orthogonality.png

The fluorescence under the test with 3 different orthogonal RBS and 3 different orthogonal ribosmes. It shows that the orthogonality of A2 ribosome and A2 RBS is one of the best ones. [[File:Shanghaitech-A2.png|450px|centerFig. 3
A2 O-16S fluorescence at 200 minutes, showing that the exist of A2 orthogonal 16s rRNA will greatly activate the GFP. However, it will be deeply depressed without the A2 ribosome. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 798
    Illegal NheI site found at 821
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1660
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 707