Difference between revisions of "Part:BBa K3866014"

(→‎Exerimental Use and Experience)
(→‎Exerimental Use and Experience)
 
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===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--GRgel.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut) Restriction digestion a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminato (C1a-C4b) with : BamHI(C1a-C4a) , Expected bands : 2847+2225 bp , EcoRV (C2a-C2b) ,Expected bands : 3587 bp + 2845 bp, Positive result: C1,C2,C3,C3 (C1a and C1b is the same sample etc)</i>]]
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[[File:T--Thessaly--GRgel.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut)Positive clones 4, 5, 6
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Expected bands : 3366 bp, 3238 bp, 2847 bp, 2194 bp, 731 bp, 197 bp
 +
</i>]]
  
 
===Exerimental Use and Experience===
 
===Exerimental Use and Experience===
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t=0 is considered when SCFAs are added.
 
t=0 is considered when SCFAs are added.
  
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*The positive control is  the TU with ECFP only <bbpart>BBa_K3505032</bbpart>
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*The positive control is  the TU with ECFP only <bbpart>BBa_K3505032</bbpart> (LacO)
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*The negative controls are the empty vector <bbpart>BBa_K3505008</bbpart>with and without SCFAs.
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*The negative controls are the empty vector <bbpart>BBa_K3505008</bbpart>with and without SCFAs.(a1R)
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*The lines in between are TANGO modules with 0, 0,1, 1 and 10 mM Sodium Acetate
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*The lines in between are TANGO modules with 0, 0,1, 1 and 10 mM Sodium Acetate(GPCR)
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[[File:T--Thessaly--GRdiag.png|700px|thumb|none|<i><b>Fig.3:</b>: TANGO assay </i>]]
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[[File:T--Thessaly--GRRFU.png|700px|thumb|none|<i><b>Fig.3:</b>: TANGO assay RFU </i>]]
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[[File:T--Thessaly--GROD.png|700px|thumb|none|<i><b>Fig.4:</b>: TANGO assay OD600 </i>]]
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[[File:T--Thessaly--GRRATIO1.png|700px|thumb|none|<i><b>Fig.5:</b>: TANGO assay Ratio RFU/OD600 </i>]]
 
<b>Conclusion </b>
 
<b>Conclusion </b>
 
<p>We observe that there was continuous non-specific suppression, both in the presence and absence of SCFAs.  
 
<p>We observe that there was continuous non-specific suppression, both in the presence and absence of SCFAs.  

Latest revision as of 17:49, 4 October 2021


TANGO module NOT GATE SCFAs detection

Usage and Biology

This composite part constitutes the G-protein coupled bioreceptor composite of the dual TANGO-GPCR assay(Dogra, Sona, Kumar and Yadav, 2016) (the other part is the b-arrestin-2 constituent, placed under the control of the arabinosed-induced promoter. FFAR2 is a naturally found eukaryotic GPCR protein that exhibits high affinity for SCFAs (acetic, propionic and butyric acid)(Kaemmerer, 2010) . After background searching through the NCBI database, we have identified the gene coding sequences needed for the designing of this gene construct. More specifically, a vasopressin receptor 2 segment has been attached to the C-terminal of the receptor, as it mediates recruitment of a TEV tagged b-arrestin-2, along with a TEV cleavage site. When TEV protease cleaves , the lac repressor is released and binds to the lac operator . In the presence of SCFAs the GPCR is activated.

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 cloned in seva alpha1R vector BBa_K3505008and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level a final module of GPCR-Tango Module.

Verification of Cloning

Fig.2:: (U=Uncut , C= Cut)Positive clones 4, 5, 6 Expected bands : 3366 bp, 3238 bp, 2847 bp, 2194 bp, 731 bp, 197 bp

Exerimental Use and Experience

This TANGO NOT GATE after SCFAs induction should lower the Fluorecence.

Cultures grown in LB and induced with 1% L-arabinose for 3 hours. t=0 is considered when SCFAs are added.

  • The positive control is the TU with ECFP only BBa_K3505032 (LacO)
  • The negative controls are the empty vector BBa_K3505008with and without SCFAs.(a1R)
  • The lines in between are TANGO modules with 0, 0,1, 1 and 10 mM Sodium Acetate(GPCR)
Fig.3:: TANGO assay RFU
Fig.4:: TANGO assay OD600
Fig.5:: TANGO assay Ratio RFU/OD600

Conclusion

We observe that there was continuous non-specific suppression, both in the presence and absence of SCFAs. We hypothesize that 1. either suppression takes place without the release of the LacI from the GPCR before it reaches the membrane, or 2. the TEV protease cleaves the LacI non-specifically.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 8313
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
    Illegal PstI site found at 7005
    Illegal PstI site found at 7092
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 8817
    Illegal NheI site found at 8840
    Illegal SpeI site found at 8313
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
    Illegal PstI site found at 7005
    Illegal PstI site found at 7092
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 6905
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 3028
    Illegal BamHI site found at 3229
    Illegal BamHI site found at 6607
    Illegal XhoI site found at 7302
    Illegal XhoI site found at 7696
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 8313
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
    Illegal PstI site found at 7005
    Illegal PstI site found at 7092
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 8313
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
    Illegal PstI site found at 7005
    Illegal PstI site found at 7092
    Illegal NgoMIV site found at 3487
    Illegal AgeI site found at 983
    Illegal AgeI site found at 2863
    Illegal AgeI site found at 6442
    Illegal AgeI site found at 7288
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI site found at 2845
    Illegal SapI site found at 3683
    Illegal SapI site found at 3938
    Illegal SapI site found at 6424
    Illegal SapI.rc site found at 3140
    Illegal SapI.rc site found at 8247
    Illegal SapI.rc site found at 8595


References

  • Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.
  • Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.