Difference between revisions of "Part:BBa K3505032"

(Extra Engineering Use)
(Extra Engineering Use)
 
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===Extra Engineering Use===
 
===Extra Engineering Use===
 
This part ,AndersonJ23114:LacO:RBS-ECFP-Double terminator , is very important for our engineering success , as it is our reporter module that is regulated from LacI. In the presence of LacI the transcription unit is blocked and there is no-fluoresence signal. This is the visualisation behind the NOT-Gate device. When we completed the cloning experiments, we immediately started plate-reader assays in order to validate the expression of ECFP , in absence of LacI. This chart shows the expression of ECFP, after 16h of incubation at 37oC , using  M9 Medium.
 
This part ,AndersonJ23114:LacO:RBS-ECFP-Double terminator , is very important for our engineering success , as it is our reporter module that is regulated from LacI. In the presence of LacI the transcription unit is blocked and there is no-fluoresence signal. This is the visualisation behind the NOT-Gate device. When we completed the cloning experiments, we immediately started plate-reader assays in order to validate the expression of ECFP , in absence of LacI. This chart shows the expression of ECFP, after 16h of incubation at 37oC , using  M9 Medium.
[[File:T--Thessaly--fluo-te-le.png|700px|thumb|none|<i><b>Fig.3:</b>Validation that J23115-TetO <bbpart>BBa_K3505044</bbpart> and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using  M9 Medium.</i>]]
+
<p>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
 +
 
 +
[[File:T--Thessaly--fluo-te-le.png|700px|thumb|none|<i><b>Fig.4:</b>Validation that J23115-TetO <bbpart>BBa_K3505044</bbpart> and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using  M9 Medium.</i>]]
  
  
 
We combined this part with <bbpart>BBa_K3505027</bbpart> a LacI regulated by an inducible promoter of SCFAs. Adding SCFAs ,LacI is expressed and the transcription unit of ECFP is repressed.
 
We combined this part with <bbpart>BBa_K3505027</bbpart> a LacI regulated by an inducible promoter of SCFAs. Adding SCFAs ,LacI is expressed and the transcription unit of ECFP is repressed.
 
<p>We characterised the part <bbpart>BBa_K2924016</bbpart>, a SCFAs' inducible promoter Flic and concluded that the most optimal concentration of SCFAs is 2mM, so we simulated the proof of concept of our project adding 2mM of SCFAs , as shown below.
 
<p>We characterised the part <bbpart>BBa_K2924016</bbpart>, a SCFAs' inducible promoter Flic and concluded that the most optimal concentration of SCFAs is 2mM, so we simulated the proof of concept of our project adding 2mM of SCFAs , as shown below.
[[File:T--Thessaly--p1.png|700px|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--p1.png|700px|thumb|none|<i><b>Fig.5:</b>NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM acetate.</i>]]
  
[[File:T--Thessaly--p3.png|700px|thumb|none|<i><b>Fig.5:</b>NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.</i>]]
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[[File:T--Thessaly--p3.png|700px|thumb|none|<i><b>Fig.6:</b>NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.</i>]]
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3505032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3505032 SequenceAndFeatures</partinfo>

Latest revision as of 00:50, 28 October 2020


pAndersonJ23115:lacO:RBS-eCFP -terminator


eCFP BBa_K3505020uder control of a constitutive promoter (andersonJ23115 with a lac operator) BBa_K2924013.

Fig.1:J23115-eCFP-Terminator

Usage and Biology

This Trancriscription Unit (TU) is continuesly activated exressing the eCFP protein as a reporter. The Lac operator that is downsteam the anderson exists for the lac regulated inhibition.


Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in both a1R BBa_K3505008and a2 BBa_K3505009 and has overhangs compatible for GoldenBraid cloning.

Verification of cloning

Cloned in alpha 2

Fig.2:(U=Uncut C=Cut) (U=Uncut , C= Cut) Restriction digestion of LE: AndersonJ23115:Lac0-EGFP-double terminator(C1-C 4) with : HindIII + BtgZI (C1-C4) , Expected bands : 3200+ 597bp ,Positive result: C1,C2

Cloned in alpha 1R

Fig.3:U=Uncut , C= Cut) Restriction digestion of AndersonJ23115:LacO -ECFP -Double Terminator (C1-C 4), with : BamHI (C1-C4) , Expected bands:2847 +952 bp. Positive result: C1,C2,C3,C4.

Experimental Use and Experinece

This part is used in BBa_K3505036


Extra Engineering Use

This part ,AndersonJ23114:LacO:RBS-ECFP-Double terminator , is very important for our engineering success , as it is our reporter module that is regulated from LacI. In the presence of LacI the transcription unit is blocked and there is no-fluoresence signal. This is the visualisation behind the NOT-Gate device. When we completed the cloning experiments, we immediately started plate-reader assays in order to validate the expression of ECFP , in absence of LacI. This chart shows the expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.

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:Validation that J23115-TetO BBa_K3505044 and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.


We combined this part with BBa_K3505027 a LacI regulated by an inducible promoter of SCFAs. Adding SCFAs ,LacI is expressed and the transcription unit of ECFP is repressed. <p>We characterised the part BBa_K2924016, a SCFAs' inducible promoter Flic and concluded that the most optimal concentration of SCFAs is 2mM, so we simulated the proof of concept of our project adding 2mM of SCFAs , as shown below.

Fig.5:NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM acetate.
Fig.6:NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]