Difference between revisions of "Part:BBa K3654015"

 
 
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This composite part is composed of two composite parts. One is the PlacI-RBS-mprA-T7 terminator (BBa_K3654012), which is responsible for the costitutively expression of mprA, and the other is the PT7-LacO-RBS-CouP-T7 terminator (BBa_K3654014), which is responsible for the IPTG inducible expression of CouP.
 
This composite part is composed of two composite parts. One is the PlacI-RBS-mprA-T7 terminator (BBa_K3654012), which is responsible for the costitutively expression of mprA, and the other is the PT7-LacO-RBS-CouP-T7 terminator (BBa_K3654014), which is responsible for the IPTG inducible expression of CouP.
  
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===Usage and Biology===
 
===Usage and Biology===
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==Biosensor performance improvement==
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Previous studies have shown that CouP, an active transporter of phenolic compounds, is involved in vanillin uptake and further enhances the conversion of vanillic acid and catechol. So, we added CouP to improve the efficacy of the 1st biosensor. We designed the expression of CouP in two methods (constitutive or inducible expression) as shown in Fig. 3.
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https://static.igem.org/mediawiki/parts/thumb/b/b6/T--KSA_KOREA--biosensor_results-fig03.png/800px-T--KSA_KOREA--biosensor_results-fig03.png
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FIGURE  3. EXPRESSION OF COUP FOR EFFECTIVE TRANSFER OF PHENOLIC COMPOUNDS
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1st Iteration
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First, to maintain the high level of CouP protein, we manufactured plasmid in a way that can be controlled by the T7 promoter and the lac operator. We prepared the transformation E. coli with the 2nd Biosensor plasmid in the same manner and dispensed IPTG according to concentration and incubated for 8 hours. Unlike our expectations, the sample in which IPTG was dispensed showed less fluorescence in contrast to that in which IPTG was not dispensed (Fig. 4).
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https://static.igem.org/mediawiki/parts/2/28/T--KSA_KOREA--biosensor_results-fig04.png
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FIGURE  4. DIFFERENCES IN BIOSENSOR FUNCTION ACCORDING TO COUP OVEREXPRESSION
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2nd Iteration
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As the overexpression of CouP could affect the expression of the Reporter gene, we constructed a new plasmid DNA in which LacI promoter controls the CouP expression,. We followed the same procedure as the previous experiments, and observed brighter red fluorescence when CouP was present. After digitizing the pixel intensity of the fluorescence with the help of ImageJ, we found that the fluorescence was about 20% higher when CouP was present (Fig. 5).
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https://static.igem.org/mediawiki/parts/4/48/T--KSA_KOREA--biosensor_results-fig05.png
  
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FIGURE  5. IMPROVING THE PERFORMANCE OF BIOSENSORS BY CONSTITUTIVELY EXPRESSED COUP
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:27, 28 October 2020


PlacI-RBS-mprA-T7 terminator - PT7-LacO-RBS-CouP-T7 terminator

This composite part is composed of two composite parts. One is the PlacI-RBS-mprA-T7 terminator (BBa_K3654012), which is responsible for the costitutively expression of mprA, and the other is the PT7-LacO-RBS-CouP-T7 terminator (BBa_K3654014), which is responsible for the IPTG inducible expression of CouP.


Usage and Biology

Biosensor performance improvement

Previous studies have shown that CouP, an active transporter of phenolic compounds, is involved in vanillin uptake and further enhances the conversion of vanillic acid and catechol. So, we added CouP to improve the efficacy of the 1st biosensor. We designed the expression of CouP in two methods (constitutive or inducible expression) as shown in Fig. 3.

800px-T--KSA_KOREA--biosensor_results-fig03.png

FIGURE 3. EXPRESSION OF COUP FOR EFFECTIVE TRANSFER OF PHENOLIC COMPOUNDS

1st Iteration First, to maintain the high level of CouP protein, we manufactured plasmid in a way that can be controlled by the T7 promoter and the lac operator. We prepared the transformation E. coli with the 2nd Biosensor plasmid in the same manner and dispensed IPTG according to concentration and incubated for 8 hours. Unlike our expectations, the sample in which IPTG was dispensed showed less fluorescence in contrast to that in which IPTG was not dispensed (Fig. 4).

T--KSA_KOREA--biosensor_results-fig04.png

FIGURE 4. DIFFERENCES IN BIOSENSOR FUNCTION ACCORDING TO COUP OVEREXPRESSION

2nd Iteration As the overexpression of CouP could affect the expression of the Reporter gene, we constructed a new plasmid DNA in which LacI promoter controls the CouP expression,. We followed the same procedure as the previous experiments, and observed brighter red fluorescence when CouP was present. After digitizing the pixel intensity of the fluorescence with the help of ImageJ, we found that the fluorescence was about 20% higher when CouP was present (Fig. 5).

T--KSA_KOREA--biosensor_results-fig05.png

FIGURE 5. IMPROVING THE PERFORMANCE OF BIOSENSORS BY CONSTITUTIVELY EXPRESSED COUP Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 115
    Illegal PstI site found at 1087
    Illegal PstI site found at 1525
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1087
    Illegal PstI site found at 1525
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1670
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 115
    Illegal PstI site found at 1087
    Illegal PstI site found at 1525
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 115
    Illegal PstI site found at 1087
    Illegal PstI site found at 1525
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1428