Difference between revisions of "Part:BBa K3505032"
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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 14h 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.</i>]] | [[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.</i>]] | ||
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+ | 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. | ||
[[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>]] | [[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>]] | ||
[[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>]] | [[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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===Sequence and Features=== | ===Sequence and Features=== | ||
<partinfo>BBa_K3505032 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3505032 SequenceAndFeatures</partinfo> |
Revision as of 14:22, 27 October 2020
pAndersonJ23115:lacO:RBS-eCFP -terminator
eCFP BBa_K3505020uder control of a constitutive promoter (andersonJ23115 with a lac operator) BBa_K2924013.
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 Golden Braid cloning.
Verification of cloning
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 14h 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]