Difference between revisions of "Part:BBa K2036019"
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| − | [[File:T--HUST-China--CI-pR_inhibition.png|thumb|800px|center| | + | [[File:T--HUST-China--CI-pR_inhibition.png|thumb|800px|center|Fig3: Cro and pRM inhibition test]] |
<h3>Preliminary experiments of LVAssrA-tag</h3> | <h3>Preliminary experiments of LVAssrA-tag</h3> | ||
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| − | [[File:T--HUST-China--Experiments-LVAssrA.png|thumb|800px|center| | + | [[File:T--HUST-China--Experiments-LVAssrA.png|thumb|800px|center|Fig4: LVAssrAtag degradation rate measurement under plac]] |
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From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein. | From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein. | ||
Latest revision as of 06:57, 25 October 2016
RFP-LAAtag-TT-pRM-RBS-GFP-LVAssrAtag
This is a part of Prokaryote version of Signal Filter ([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K2036027).
Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.
We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.
Preliminary experiments of LVAssrA-tag
In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery ( BBa_J04500) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.
From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 573
Illegal AgeI site found at 685 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1601