Part:BBa_K3602020:Experience

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Applications of BBa_K3602020

Protein purification and His-tag removal

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

Figure 1. Successful protein purification of Cas13a. The first lane displays a sample of lysed E. coli ‘ER2536+ Cas13a plasmid’ flow-through before induction with IPTG. The second lane displays a sample of lysed E. coli ‘ER2536 + Cas13a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The fourth lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lane 7-12 displays 1 mL elution-buffer fractions.In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas13a+SUMO-Histag = 153,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.

The protein concentration was estimated with the Bradford assay. Concentrations for Cas13a at a range 50-80 ng/mL for fraction 8-11 were achieved. Lane 2 is lysate before IPTG-induction and lane 3 is lysate after IPTG-induction.

A sumo protease was used to cleave 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.

Figure 2. Successful cleaving of SUMO-6x His-tag from Cas13a. 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, a band is seen at around 140 kDa, fitting relatively well with the cleaving of SUMO-6Xhis-tag, isolation a Cas13a with the molecular weight of 148,8 kD.a 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 1. Proof of Cas13a collateral cleavage being dependent on sgRNA concentration. All samples contain 20 ng/µL rRNA as a marker. Lane 1 and 2 are the positive and negative controls receptively. The positive control contains 1 µg/µL RNase A and the negative only contains rRNA and nuclease-free water. Lane 3-6 were incubated in 15 mins at 37°C, lane 7-19 were incubated for 40 mins and lane 11-14 were incubated for 3 hours. For each time samples with four different sgRNA concentrations were used. Thus, the samples contained 35 ng/µL Cas13a, 0.43 ng/µL target and 1.7 ng/µL. The varying concentration of sgRNA are lane 3, 7, and 11, = 0.43 ng/µL for lane 4, 8, and 12 = 0.043 ng/µL for lane 5, 9, and 13, = for lane 6, 10, and 14 = 0.0043 ng/µL.

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 3 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.

Figure 4. Proof of Cas13a collateral cleavage being dependent on sgRNA concentration. All samples contain 20 ng/µL rRNA as a marker. Lane 1 and 2 are the positive and negative controls receptively. The positive control contains 1 µg/µL RNase A and the negative only contains rRNA and nuclease-free water. Lane 3-6 were incubated in 15 mins at 37°C, lane 7-19 were incubated for 40 mins and lane 11-14 were incubated for 3 hours. For each time samples with four different sgRNA concentrations were used. Thus, the samples contained 35 ng/µL Cas13a, 0.43 ng/µL target and 1.7 ng/µL. The varying concentration of sgRNA are lane 3, 7, and 11, = 0.43 ng/µL for lane 4, 8, and 12 = 0.043 ng/µL for lane 5, 9, and 13, = for lane 6, 10, and 14 = 0.0043 ng/µL.

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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