Team:NTU SINGAPORE/Collaborations



Collaboration

Collaborating with these teams allowed us to achieve our goals. It was our pleasure to help them to the best of our ability.

Team Macquarie



How did we help them?


Team Macquarie had constructed a hydrogenase gene capable of converting glucose to hydrogen gas. The plasmid BBa_K2300001 consists of the genes for hydrogenase enzyme (Hyd1), ferredoxin, ferredoxin-NADPH-reductase (FNR) and maturation enzyme (HydEF and HydG). Their expression levels are co-regulated under the pLac promoter.


This year, our team helped Team Macquarie to evaluate each of the gene expression levels using RT-PCR.


The results of RT-PCR are shown 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 were used to induce gene expression. Comparison of 1mM IPTG and 20mM IPTG was made and it was found that 1mM IPTG has a higher gene expression level. It is worth noting that the RT-PCR efficiency might be different for different genes due to varied sizes of PCR products and the formula used above assumes that the PCR efficiency is 100% such that 2 copies of products are eventually obtained.

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 each of the various dCas9 mutants with its respective truncations based on our design, suggesting that the expression and purification procedure were performing well.


To test the dCas9 constructs, Team Macquarie adapted their in vitro CRISPR assay from the previous year to include an electrophoretic migration shift assay. As the dCas9 constructs were designed to lack nuclease activity, whilst retaining the ability to bind to the gRNA and target DNA, variations of the in vitro CRISPR assay were set up 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). This was compared 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.




The experiment used a commercial Cas9 with nuclease activity as a control (see Fig. 2). From this sample, we have been able to better interpret our results. The pre-incubated control shows an upward gel shift of the gRNA (see Fig. 2A), indicating commercial Cas9 binding to gRNA. Upon incubation with the PCR product, there is a disappearance of both higher bands and loss of smearing, suggesting the formation of a stable conformation of gRNA, Cas9 and the target PCR product. The deactivation step causes the disassembly of the gRNA/Cas9 complex, resulting in the reverting of the gel shift of the PCR product and the gRNA, and also shows specific cleavage of the PCR product. Absence of the protein bands after deactivation is likely due to denaturation of the enzyme that results in protein aggregation/precipitation.

From their results, different truncated protein sites in the four samples were observed: WT (dCas9), 3P, 5P and HNH (see Fig. 1). The EMSA performed suggests binding to gRNA of mutant dCas9 in WT, 3P, 5P and HNH samples. This is evident from the observation of gRNA complexes shifting upon incubation with DNA. Protein shifts after incubation with the PCR product suggested that the mutants retained the ability to bind the target DNA. Deactivation of the protein DNA complex reversed the migration shift of the PCR product, providing further evidence that the dCas9 binds to the PCR product, while not having nuclease activity.





Team NUS



We helped the Team NUS to characterize their plasmid BBa_K2447014. The protocol can be found here

However, the results we obtained are not consistent with their expectations.