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

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<h2>Results</h2>
 
<h2>Results</h2>
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<p>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.</p>
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<p>In the EMSA gel experiment, Macquarie utilized the considerably purer commercial WT Cas9 to provide a reference point as to identity of bands. They confirmed that our WT dCas9 and all truncation variants were unable to cleave target DNA, as evidenced by singular uncut ferrochelatase PCR product in lane B9, D5, D8 and D11, compared to the cut products for commercial WT-Cas9 in lane B5.</p>
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<p>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.</p>
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<p>Mobility shift was apparent for our WT-dCas, when comparing the band size of ferrochelatase PCR target between lane 8 and 9, and this was reflected in commercial WT Cas9, albeit fainter due to most undergoing cleavage and dissociating. This shift appears to be due to gRNA binding, as band is observed in the RNP lane with only dCas9 and gRNA. DNA binding appears to result in a very large RNPF complex that is unable to enter the gel, as evidenced by the bright DNA band in the well of B4. The same shift did not seem to appear with our WT-dCas9, although a high MW smear was observed. This phenomenon seems isolated as the same smear was not observed with the truncated dCas9 variants. </p>
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<p>They demonstrated liberation of gRNA upon denaturation in Figure B lane B5. Unfortunately, the same gRNA bands were not observed in our wild-type dCas9. Similar gRNA bands were observed for truncated dCas9 in figure D. Based on difference in intensity of the gRNA bands between RNP/RNPF lanes and denatured lane, results seems to suggest that HNH is able to bind to gRNA best, resulting is lesser free gRNA, followed by 3ple and 5ple. This corresponds to our gene activation tests, confirming that the poorer gene activation observe is not just due to poorer target DNA binding – it is likely that poor gRNA binding could be a contributing factor for the poor gene activation by truncated dCas9-VPR. </p>
 +
<p>The lack of supershifted RNFP in WT-dCas9 experiments coupled with a high MW smear led us to suspect potential DNase/RNase contamination. Such a contamination would also explain the loss of gRNA band in the denaturation lane. Supershifted RNFP also adds doubt to intensity of RNPF complex band. A lower percent native gel and a smaller target DNA PCR product would likely result in a smaller shift, and be more in line to established dCas9 EMSA experiments in literature. Noting these weaknesses, we look forward to further improving this protocol in the future. Such information would conclusively characterize the altered binding affinities for target DNA and gRNA by our truncated dCas9 variants. </p>
 +
<p>We are deeply thankful to Macquarie iGEM team for going above and beyond in this iGEM collaboration. We had initially proposed this experiment to merely observe DNA binding causing a supershift, but the results has taught us much more – such as about the variable gRNA binding by truncated dCas9. A special shoutout to Dr Edward Moh, whose expertise has made the planning of this collaboration a fun process, despite us not being familiar with the experiment. </p>
 
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Revision as of 13:28, 1 November 2017



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Collaboration

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


NUS Singapore
Macquarie Australia

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?


In our gene activation experiments, we observed that our truncated dCas9 generally exhibit poorer gene activation compared to full length wild type. We had hypothesized that this is likely due to poorer target DNA binding, and hence shorter dwell time at the promoter for the VPR transcriptional activator to work effectively. One key weakness with our experiments was that we were unable to directly characterize target DNA binding affinity by our truncated dCas9 variants.


For our collaboration with Macquarie iGEM team, we had a serendipitous meet-up with their advisor in Singapore. Noting their protein purification capabilities, we suggested an EMSA affinity binding assay. EMSA affinity binding assay is a well-documented method to characterize dCas9 binding to target DNA. Native gel PAGE is utilized, whereupon dCas9 binding to target DNA, a mobility shift upwards is observed due to increased size, and hence decreased migration. The intensity of the shifted band would indicate amount of dCas9 binding to target DNA. We hypothesize that truncated dCas9 would have decreased binding, and hence lower band intensity.


Our dCas9 wild type and truncated variants were cloned into BPK 65767 (gift from Keith Joung Lab, Addgene) with 6x His tag, using Gibson assembly. The original Cas9 is replaced – this is necessary as we are investigating binding without cutting with dCas9, and all dCas9 proteins were human codon optimized. Macquarie iGEM team utilized commercially synthesized sgRNA that they have previously shown to work with Cas9.

Results


In the EMSA gel experiment, Macquarie utilized the considerably purer commercial WT Cas9 to provide a reference point as to identity of bands. They confirmed that our WT dCas9 and all truncation variants were unable to cleave target DNA, as evidenced by singular uncut ferrochelatase PCR product in lane B9, D5, D8 and D11, compared to the cut products for commercial WT-Cas9 in lane B5.

Mobility shift was apparent for our WT-dCas, when comparing the band size of ferrochelatase PCR target between lane 8 and 9, and this was reflected in commercial WT Cas9, albeit fainter due to most undergoing cleavage and dissociating. This shift appears to be due to gRNA binding, as band is observed in the RNP lane with only dCas9 and gRNA. DNA binding appears to result in a very large RNPF complex that is unable to enter the gel, as evidenced by the bright DNA band in the well of B4. The same shift did not seem to appear with our WT-dCas9, although a high MW smear was observed. This phenomenon seems isolated as the same smear was not observed with the truncated dCas9 variants.

They demonstrated liberation of gRNA upon denaturation in Figure B lane B5. Unfortunately, the same gRNA bands were not observed in our wild-type dCas9. Similar gRNA bands were observed for truncated dCas9 in figure D. Based on difference in intensity of the gRNA bands between RNP/RNPF lanes and denatured lane, results seems to suggest that HNH is able to bind to gRNA best, resulting is lesser free gRNA, followed by 3ple and 5ple. This corresponds to our gene activation tests, confirming that the poorer gene activation observe is not just due to poorer target DNA binding – it is likely that poor gRNA binding could be a contributing factor for the poor gene activation by truncated dCas9-VPR.

The lack of supershifted RNFP in WT-dCas9 experiments coupled with a high MW smear led us to suspect potential DNase/RNase contamination. Such a contamination would also explain the loss of gRNA band in the denaturation lane. Supershifted RNFP also adds doubt to intensity of RNPF complex band. A lower percent native gel and a smaller target DNA PCR product would likely result in a smaller shift, and be more in line to established dCas9 EMSA experiments in literature. Noting these weaknesses, we look forward to further improving this protocol in the future. Such information would conclusively characterize the altered binding affinities for target DNA and gRNA by our truncated dCas9 variants.

We are deeply thankful to Macquarie iGEM team for going above and beyond in this iGEM collaboration. We had initially proposed this experiment to merely observe DNA binding causing a supershift, but the results has taught us much more – such as about the variable gRNA binding by truncated dCas9. A special shoutout to Dr Edward Moh, whose expertise has made the planning of this collaboration a fun process, despite us not being familiar with the experiment.





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.