Difference between revisions of "Part:BBa K2819000"
 
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<b>Real-time Polymerase Chain Reaction (qPCR)</b><br>
 
<b>Real-time Polymerase Chain Reaction (qPCR)</b><br>
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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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[http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2819200 BBa_K2819200], which contains this coding region, for us as part of our collaboration. BL21 (DE3) carrying this plasmid was induced according to our protocol. <br>
  
 
<u>Exponential Fold Change was calculated according to the following formula: </u><br>
 
<u>Exponential Fold Change was calculated according to the following formula: </u><br>
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Exponential fold of gene = 2<sup>-(ΔΔCt)</sup><br><br>
 
Exponential fold of gene = 2<sup>-(ΔΔCt)</sup><br><br>
  
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[[File:T--NUS_Singapore-A--aTc_Concentration.jpg|500px|center|]]
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[[File:T--NUS_Singapore-A--Results_qPCR_F3%27H_Brep.jpg|500px|center|]]
 
<font size="2"><center><b>Figure 1: Exponential Fold Change of <i>F3'H</i> gene </b></center></font><br><br>
 
<font size="2"><center><b>Figure 1: Exponential Fold Change of <i>F3'H</i> gene </b></center></font><br><br>
  
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Figure 1 shows that the mRNA of FNS is transcribed, suggesting that our part is working as intended. Induction was carried out in dark, hence, P<sub>BLrep</sub> would behave like a constitutive promoter. However, exponential fold change of the F3’H gene seems to increase in higher aTc concentration, when it should not be affected by the inducer concentration. This suggests that further optimization is required for this part.<br><br>
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Figure 1 shows that the mRNA of F3'H is transcribed, suggesting that our part is working as intended. Induction was carried out in dark, hence, P<sub>BLrep</sub> would behave like a constitutive promoter. However, exponential fold change of the F3’H gene seemed to increase in higher aTc concentration, when it should not be affected by the inducer concentration. This suggests that further optimization is required for this part.<br><br>
  
 
<b>Biosynthesis of Luteolin</b><br>
 
<b>Biosynthesis of Luteolin</b><br>
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===Conclusion===
 
===Conclusion===
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The above characterisation results indicate that the <i>F3'H</i> gene is being produced, however, further optimization is needed to better understand the gene.<br>
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As both F3'H and FNS are needed in the biosynthesis of luteolin, these characterisation results indicate that the <i>F3'H</i> gene is being produced, however, further optimization is needed to better understand the gene.<br>
  
  

Latest revision as of 14:29, 17 October 2018


Coding Region for F3'H

This part is the coding sequence of flavonoid 3′-hydroxylase (F3′H). This gene comes from Gerbera hybrida, and the sequence was obtained after codon optimization.

Usage and Biology

- This engineered part can be used to produce Eriodictyol, a flavonoid.
- To achieve optimal expression of this coding region, our modelling results suggested to use a strong promoter as its Kcat value is low.
- In addition, we recommend BL21* (DE3) as a chassis for this composite part. This is because BL21* (DE3) is an RNase knockout strain, therefore the half-life of mRNA is prolonged in this strain, which can help in the biomanufacturing of the E. coli.

Characterization

To characterize this part, we expressed it under an EL222 blue light repressible promoter, BBa_K2332019. To obtain the sequence of this composite part, refer to BBa_K2819200.

Real-time Polymerase Chain Reaction (qPCR)
[http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on BBa_K2819200, which contains this coding region, for us 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(wild type) - Ct (16S gene)
ΔCt1 = Ct(cell with genes) - Ct (16S gene)
ΔΔCt = ΔCt2 - ΔCt1
Exponential fold of gene = 2-(ΔΔCt)

T--NUS Singapore-A--Results qPCR F3'H Brep.jpg
Figure 1: Exponential Fold Change of F3'H gene


Figure 1 shows that the mRNA of F3'H is transcribed, suggesting that our part is working as intended. Induction was carried out in dark, hence, PBLrep would behave like a constitutive promoter. However, exponential fold change of the F3’H gene seemed to increase in higher aTc concentration, when it should not be affected by the inducer concentration. This suggests that further optimization is required for this part.

Biosynthesis of Luteolin
F3’H is involved in the luteolin-synthesis pathway. We use co-transformed this part with BBa_K2819206 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


Conclusion

As both F3'H and FNS are needed in the biosynthesis of luteolin, these characterisation results indicate that the F3'H gene is being produced, however, further optimization is needed to better understand the gene.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 713
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]