Difference between revisions of "Part:BBa K2819206"

 
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<partinfo>BBa_K2819206 short</partinfo>
 
<partinfo>BBa_K2819206 short</partinfo>
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This part contains the coding sequence of Flavone Synthase (FNS), inducible by arabinose. This gene comes from <i>Petroselinum crispum</i>, and the sequence is obtained after codon optimization.
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===Characterization===
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<b>Real-time Polymerase Chain Reaction (qPCR)</b>
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[http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on our composite parts as part of our collaboration. BL21 (DE3) carrying this plasmid was induced according to our protocol. <br>
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Exponential Fold Change was calculated according to the following formula: <br>
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ΔCt1= Ct(empty cells)- Ct(16S gene)<br>
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ΔCt1 = Ct(cell with genes)-Ct (16S gene)<br>
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ΔΔCt = ΔCt2 - ΔCt1 <br>
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Exponential fold of gene = 2<sup>-(ΔΔCt)</sup><br>
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[[File:T--NUS_Singapore-A--Arabinose_Concentration.jpg|500px|center|]]
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<font size="1"><center><b>Figure 1: Exponential Fold Change of <i>FNS</i> gene </b></center></font><br><br>
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As seen from figure 1, the mRNA of FNS is transcribed, this suggests that our part is working as intended. However, mRNA seems to be transcribed even without arabinose, indicating that PBAD is leaky. However, the expression level of FNS without arabinose seems to be higher or equal to than when 10mM and 20mM of arabinose are present. This suggests that further optimization is required for this part. <br>
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<b>Biosynthesis of Luteolin</b>
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FNS is involved in the luteolin-synthesis pathway. We use co-transformed this part with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2819200 BBa_K2819200] into BL21* (DE3) for the biosynthesis of luteolin. We not only carried out the biosynthesis in flasks, but also in a bioreactor.
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[[File:T--NUS_Singapore-A--Luteolin_bioreactor.jpg|400px|center|]]
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<font size="1"><center><b>Figure 2: Luteolin in the supernatant of the cell cultures from biosynthesis using a bioreactor </b></center></font><br><br>
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<b>High Performance Liquid Chromatography (HPLC)</b><br>
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To confirm that we have produced luteolin, we carried out HPLC, and the following shows the results. <br>
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[[File:T--NUS_Singapore-A--Luteolin_Biosynthesis_conc_new_part.jpg|500px|center|]]
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<font size="1"><center><b>Figure 3: Comparison of Luteolin concentration in BL21* (DE3) wild type and BL21* (DE3) with our parts </b></center></font><br><br>
  
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:15, 15 October 2018


Arabinose Inducible Production of Flavone Synthase (FNS) This part contains the coding sequence of Flavone Synthase (FNS), inducible by arabinose. This gene comes from Petroselinum crispum, and the sequence is obtained after codon optimization.

Characterization

Real-time Polymerase Chain Reaction (qPCR) [http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on our composite parts as part of our collaboration. BL21 (DE3) carrying this plasmid was induced according to our protocol.

Exponential Fold Change was calculated according to the following formula:
ΔCt1= Ct(empty cells)- Ct(16S gene)
ΔCt1 = Ct(cell with genes)-Ct (16S gene)
ΔΔCt = ΔCt2 - ΔCt1
Exponential fold of gene = 2-(ΔΔCt)

T--NUS Singapore-A--Arabinose Concentration.jpg
Figure 1: Exponential Fold Change of FNS gene


As seen from figure 1, the mRNA of FNS is transcribed, this suggests that our part is working as intended. However, mRNA seems to be transcribed even without arabinose, indicating that PBAD is leaky. However, the expression level of FNS without arabinose seems to be higher or equal to than when 10mM and 20mM of arabinose are present. This suggests that further optimization is required for this part.

Biosynthesis of Luteolin FNS is involved in the luteolin-synthesis pathway. We use co-transformed this part with BBa_K2819200 into BL21* (DE3) for the biosynthesis of luteolin. We not only carried out the biosynthesis in flasks, but also in a bioreactor.

T--NUS Singapore-A--Luteolin bioreactor.jpg
Figure 2: Luteolin in the supernatant of the cell cultures from biosynthesis using a bioreactor


High Performance Liquid Chromatography (HPLC)
To confirm that we have produced luteolin, we carried out HPLC, and the following shows the results.

T--NUS Singapore-A--Luteolin Biosynthesis conc new part.jpg
Figure 3: Comparison of Luteolin concentration in BL21* (DE3) wild type and BL21* (DE3) with our parts



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961