Part:BBa_K3602001:Experience

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Applications of BBa_K3602001

Protein purification and His-tag removal

The His-tags are seen to work. This is seen in Figure 1 as the Cas13a protein was successfully purified from E. coli K12 using His-tag purification.

A sumo protease was used to cleaved the part to separate Cas13a from the tags. After sumo protease digestion it was shown by SDS-page that the sumo tag was successfully cleaved from the Cas13a. This is seen in Figure 2.

SUMO protease: Successful cleaving of SUMO-6xhis-tag from Cas13a Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”Gel: Bis-Tris 4-12% Running buffer:MOPS

The first lane displays Cas13a above 140 kDa as expected. The second lane displays cas13a after incubation with SUMO protease following the SUMO-protease assay from Invitrogen. The SUMO-6Xhis-tag has a molecular weight of 5 kDa. In the second lane, we see a band There is a band 140 kDa this fits relatively well with the cleaving of SUMO-6Xhis-tag, isolation a Cas13a with the molecular weight of 148,8 kDa.

With these two results, it is observed that the His-tags are working.

Cas13a function

It is seen that Cas13a is dependent on the sgRNA. In Figure 3, sgRNA (BBa_K3602004) and Cas13a are co-incubated. Then the target (BBa_K3602007) is added to the samples along with ribosomal RNA (rRNA). The samples are then incubated for 15, 40 mins or 2 hours to activate collateral cleavage of Cas13a. The activation of Cas13a is seen as a smear indicating that the Cas13a is activated and start to collaterally cleave surrounding rRNA. When comparing to the negative control (lane 2) in Figure 5, it is seen that all rRNA is degraded to some level in all other lanes. The rRNA in the positive control (lane 1) is totally degraded thereby leaving no signal. For the samples with Cas13a, sgRNA and target it is observed that the more time the samples were incubated the more rRNA is degraded. This indicates that the prolonged time gives Cas13a more time to degrade rRNA or that the rRNA is degraded because of the heat. Within the different time intervals, it is generally observed that the rRNA in the samples with 0,043 ng/µL sgRNA are most degraded ones. However, it is seen that the 0,0043 ng/µL samples are the less degraded ones besides from the negative control. This indicates, that for Cas13a to be fully activated an amount greater than 0,0043 ng/µL is needed. Thus, Cas13a is dependent on sgRNA binding upon binding to target and then activation.

Figure 4 shows that sgRNA-Cas13a complex is activated upon binding of the target sequence. The experiment is set up as the experiment in Figure 3 with sgRNA (BBa_K3602004) and target (BBa_K3602007). When the target was added to the samples, they were incubated in 40 mins. The experiment in Figure 4 showed that upon addition of target RNA to the sgRNA-Cas complex solution, collateral cleavage was induced. This can be observed as a smear in lane 5 and 6 thereby indicating degradation of rRNA. It is possible to see some degraded rRNA in all samples.

With these two results, it is shown that Cas13a can form a complex with the sgRNA. it is also observed that when a target is added to the complexes, Cas13a is activated and begin collateral cleavage. Thereby this part work as expected.

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