Difference between revisions of "Part:BBa K3505036"

 
(Experimental Use and Experience)
 
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<partinfo>BBa_K3505036 short</partinfo>
 
<partinfo>BBa_K3505036 short</partinfo>
  
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[[File:T--Thessaly--pflic.jpeg|600px|thumb|none|<i><b>Fig.1:</b> Activation of eCFP in absence of SCFAs,Based on NOT GATE</i>]]
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[[File:T--Thessaly--notgate.png|600px|thumb|none|<i><b>Fig.2:</b> The NOT GATE</i>]]
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===Usage and Biology===
 
===Usage and Biology===
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2 Trancription Units
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*LacI <bbpart>BBa_K3505003</bbpart>regulated by inducible promoter FliC <bbpart>BBa_K2924016</bbpart>which is activated from SFCAs and more specifically from Butyrate
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*eCFP <bbpart>BBa_K3505019</bbpart>under control of lac regulated Anderson<bbpart>BBa_K3505013</bbpart>.
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<b>This way we take signal in the Absence of SCFAs</b>
  
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<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3505036 SequenceAndFeatures</partinfo>
 
  
  
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===Design Notes===
===Functional Parameters===
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The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in <bbpart>BBa_K3505010</bbpart> and has overhangs compatible for GoldenBraid cloning.
<partinfo>BBa_K3505036 parameters</partinfo>
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===Verification of cloning===
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[[File:T--Thessaly--pflicfinal.png|600px|thumb|none|<i><b>Fig.3:</b>U2 C2  pFliC:RBS-LacI-Terminator-pAndersonJ23115:LacO:RBS-ECFP-terminator  EcoRV, HindIII. Expected bands 3734, 1699.</i>]]
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===Experimental Use and Experience===
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Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates
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[[File:T--Thessaly--p1.png|600px|thumb|none|<i><b>Fig.4:</b>NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM acetate.</i>]]
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[[File:T--Thessaly--p2.png|600px|thumb|none|<i><b>Fig.5:</b>Cell growth in the presence or absence of 2mM acetate.</i>]]
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[[File:T--Thessaly--p3.png|600px|thumb|none|<i><b>Fig.6:</b>NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM propionate.</i>]]
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[[File:T--Thessaly--p4.png|600px|thumb|none|<i><b>Fig.7:</b>. Cell growth in the presence or absence of 2mM propionate.</i>]]
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===Conculusion===
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There is a slight increase in fluorescence in absence of SCFAs - acetate and propionate - as expected. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. As far as the cell growth goes, we expected a reduction of the cells, since we add acids that are toxic to the cells. These results indicate that our NOT-Gate system works, regarding these acids but in an expected range.
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===Sequnce and Features===
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<partinfo>BBa_K3505036 SequenceAndFeatures</partinfo>

Latest revision as of 00:50, 28 October 2020


pFliC:RBS-LacI-Terminator- pAndersonJ23115:LacO:RBS-ECFP-terminator

Fig.1: Activation of eCFP in absence of SCFAs,Based on NOT GATE
Fig.2: The NOT GATE


Usage and Biology

2 Trancription Units

This way we take signal in the Absence of SCFAs


Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in BBa_K3505010 and has overhangs compatible for GoldenBraid cloning.

Verification of cloning

Fig.3:U2 C2 pFliC:RBS-LacI-Terminator-pAndersonJ23115:LacO:RBS-ECFP-terminator EcoRV, HindIII. Expected bands 3734, 1699.

Experimental Use and Experience

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates


Fig.4:NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM acetate.


Fig.5:Cell growth in the presence or absence of 2mM acetate.


Fig.6:NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM propionate.


Fig.7:. Cell growth in the presence or absence of 2mM propionate.

Conculusion

There is a slight increase in fluorescence in absence of SCFAs - acetate and propionate - as expected. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. As far as the cell growth goes, we expected a reduction of the cells, since we add acids that are toxic to the cells. These results indicate that our NOT-Gate system works, regarding these acids but in an expected range.

Sequnce and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1321
    Illegal NheI site found at 1344
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]