Difference between revisions of "Team:NTU SINGAPORE/Collaborations"

Macquarie iGEM Team

How did we help them?

Macquaire team had constructed a hydrogenase gene capable of converting glucose to hydrogen gas. The plasmid BBa_K2300001 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.

IPTG and 20mM of glucose was used to induce the gene expression. Comparison of 1mM IPTG and 20mM IPTG was made and it was found that 1mM IPTG has higher gene expression level. 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.

How did they help us?

Team Macquire helped us to purify dCas9 mutants and test the dCas9 constructs using an in vitro CRISPR assay.

The plasmids were transformed into BL21 (DE3) E. coli and expressed under auto-induction conditions. Cells were then harvested, lysed and purified using a Ni-NTA resin with standard His-tag purification protocols. Eluates were then run on an SDS-PAGE gel (Fig 1). The largest band in each of the eluates corresponds to the various dCas9 mutants with its respective truncations based on our design, suggested that the expression and purification procedure was performing well.

To test the dCas9 constructs, Team Macquarie adapted their in vitro CRISPR assay from the previous year to include in electrophoretic migration shift assay. As the dCas9 constructs were designed to not have nuclease activity, while retaining the ability to bind to the gRNA and target DNA, we set up variations of the in vitro CRISPR assay to track the progression of the target DNA, gRNA, cleavage products, and protein migration on a DNA retardation gel (6% acrylamide in TBE, ThermoFisher Scientific), while comparing to a commercial Cas9 with nuclease activity.

The four purified samples were subjected to three treatments:

• Pre-incubated: mix of tracrRNA and crisprRNA to form guideRNA (incubated at room temperature for 5 minutes in all samples)

• Incubated: gRNA with the addition of the Ferrocheletase PCR product (further incubated for 1 hour at 37°C)

• Deactivated: gRNA with the addition of the Ferrocheletase PCR product (further incubated for 1 hour at 37°C) and a denaturation step of 20 minutes at 80°C.

We collaborated with NUS