Difference between revisions of "Team:NTU SINGAPORE/Collaborations"
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<p>ΔCt(test)2 = Ct(Induced operon genes)-Ct(Induced Chloramphenicol gene)</p> | <p>ΔCt(test)2 = Ct(Induced operon genes)-Ct(Induced Chloramphenicol gene)</p> | ||
</br> | </br> | ||
| − | Finally, use ΔCt(test)2- ΔCt(control)1 to get ΔΔCt.</p> | + | <p>Finally, use ΔCt(test)2- ΔCt(control)1 to get ΔΔCt.</p> |
<p>5mM of IPTG and 20mM of glucose was used to induce the gene expression. It is worth to notice that the RT-PCR efficiency might be different for different genes due to their size of PCR products and the formula used above is assuming the PCR efficiency to be 100% that eventually resulted in 2 copies of products.</p> | <p>5mM of IPTG and 20mM of glucose was used to induce the gene expression. It is worth to notice that the RT-PCR efficiency might be different for different genes due to their size of PCR products and the formula used above is assuming the PCR efficiency to be 100% that eventually resulted in 2 copies of products.</p> | ||
Revision as of 12:45, 24 October 2017
Macquaire iGEM Team
Macquaire team had constructed a hydrogenase gene capable of converting glucose to hydrogen gas. The plasmid (BBa_KXXXXXX) that consist of hydrogenase enzyme (Hyd1), ferredoxin, ferredoxin-NADPH-reductase (FNR) and maturation enzyme (HydEF and HydG), in which their expression are co-regulated under pLac promoter.
This year again, our team helped them to evaluate each of the gene expression level by using RT-PCR.
The results of RT-PCR is shown as below:
Formula: 2^-(ΔΔCt) is used to calculate the fold change of gene expression after induction, in which
ΔCt(control)1= Ct(uninduced operon genes)- Ct(Chloramphenicol gene)
ΔCt(test)2 = Ct(Induced operon genes)-Ct(Induced Chloramphenicol gene)
Finally, use ΔCt(test)2- ΔCt(control)1 to get ΔΔCt.
5mM of IPTG and 20mM of glucose was used to induce the gene expression. It is worth to notice that the RT-PCR efficiency might be different for different genes due to their size of PCR products and the formula used above is assuming the PCR efficiency to be 100% that eventually resulted in 2 copies of products.
