Difference between revisions of "Part:BBa K3317007"

 
 
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<partinfo>BBa_K3317007 short</partinfo>
 
<partinfo>BBa_K3317007 short</partinfo>
  
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Sensitive promoter against protocatechuic acid (PCB)
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Sensitive promoter against protocatechuic acid and presents its maximum induction in the presence of 1000mM RPU 1.6 of vanillinic acid with a leak of 4.5x10-3  RPU in absence of induction(Meyer, A. J <i> et. al</i> 2019) this part needs its regulatory protein (<partinfo>K3317005 </partinfo>)
  
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<!-- Add more about the biology of this part here
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<!-- Add more about the biology of this part here -->
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===Usage and Biology===
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==Improve the p3B5C==
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[[Part:BBa_K3395009|p3b5b]] was obtained by point mutation of [[Part:BBa K3317007|p3b5c]],iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part [[Part:BBa K3317007|p3b5c]].
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==Characterization==
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The [[BBa_K3395015|pcauam_p3B5B]] system is constructed with two parts, upstream activator, and downstream reporter.[[Part:BBa_K3395009|p3b5b]] was obtained by point mutation of [[BBa K3317007|p3b5c]],iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part [[Part:BBa K3317007|p3b5c]]. The target of this system is to test the sensitivity of the PcauAM activator and p3b5b promoter combination. The upstream sequence is composed of  [[Part:BBa_K3395008|pLacIQ promoter]], [[Part:BBa_K3395006|PcauAM]] and [[Part:BBa_K3395014|tonB terminator]] . The upstream sequence will express PcauAM protein, which is not stable, and denature itself. The downstream sequence is composed of [[Part:BBa_K3395009|p3b5b promoter]], [[Part:BBa_K3395007|eYFP reporter]] and L3S3P21(terminator). This downstream system will present high expression when PcauAM protein is generated. We use eYFP to show the sensitivity of this combination.
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===Design===
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We constructed the promoter into the low copy p15A plasmid with YFP downstream its regulatory protein PcauAM. We transformed pET28b_p3B5B vector into <i>E .coli</i> BL21 for testing.
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[[File:T--RDFZ-China--pcauAM p3B5B.png|400px|thumb|center|Biosensor circuit of pcauam_p3B5B]]
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===Experimenting===
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Activity test:
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Sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light.
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[[File:T--RDFZ-China--sensor activity test.png|400px|thumb|center|Result of activity test]]
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The data in the right of figure are the tested data of PcaUAM-YFP under emission light 525nm.
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When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 11:26, 27 October 2020


Inducible p3B5C promoter

Sensitive promoter against protocatechuic acid and presents its maximum induction in the presence of 1000mM RPU 1.6 of vanillinic acid with a leak of 4.5x10-3 RPU in absence of induction(Meyer, A. J et. al 2019) this part needs its regulatory protein (BBa_K3317005)


Improve the p3B5C

p3b5b was obtained by point mutation of p3b5c,iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part p3b5c.

Characterization

The pcauam_p3B5B system is constructed with two parts, upstream activator, and downstream reporter.p3b5b was obtained by point mutation of p3b5c,iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part p3b5c. The target of this system is to test the sensitivity of the PcauAM activator and p3b5b promoter combination. The upstream sequence is composed of pLacIQ promoter, PcauAM and tonB terminator . The upstream sequence will express PcauAM protein, which is not stable, and denature itself. The downstream sequence is composed of p3b5b promoter, eYFP reporter and L3S3P21(terminator). This downstream system will present high expression when PcauAM protein is generated. We use eYFP to show the sensitivity of this combination.

Design

We constructed the promoter into the low copy p15A plasmid with YFP downstream its regulatory protein PcauAM. We transformed pET28b_p3B5B vector into E .coli BL21 for testing.

Biosensor circuit of pcauam_p3B5B

Experimenting

Activity test: Sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light.

Result of activity test

The data in the right of figure are the tested data of PcaUAM-YFP under emission light 525nm. When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]