Engineering

Design

To make the use of Multi Targeter closer to social implementation, we attempted to construct a system for actual use of gRNAs designed in Cycle 2.

Build

If one Cas9 plasmid is designed with the gRNA corresponding to the mutation, Cas9 must be redesigned each time the gRNA sequence changes. This method takes a long time to respond to mutations. Therefore, we designed the compatible Cas9 plasmid to allow easy introduction of various gRNAs.

Test

First, we designed a Cas9 plasmid that can easily insert various gRNA. In this plasmid, nine DNA fragments were introduced by Gibson Assembly into the backbone Cas9 plasmid. In addition, all DNA fragments and backbone Cas9 plasmid were prepared from plasmids from the iGEM Kit plate. *Figure4-1 Next, gRNA was introduced by Golden Gate assembly using Bbs1 and T4 ligase. *Figure4-2

Here is the link for experiments

Learn

This trial allowed us to build systems for effective utilization of gRNA designed with the Multi Targeter. A series of systems were constructed by creating compatible Cas9 plasmids, determining model genes, designing gRNAs, enzymatic processing, and completing Cas9 with gRNA.

Figure4-3. Engineering success

To establish a more plausible system, we need to confirm the expression and activity of the Cas9 protein in the gRNACasPlasmid that we constructed in addition to our test. For this purpose, we will perform tests to confirm whether the AmpR fragment can be cleaved in vitro, and trials to confirm protein expression by SDS-PAGE. Once it is confirmed that the Cas9 plasmid constructed by these trials is normally expressed and works normally, we will confirm the action of the Cas9 plasmid in vivo. We believe that this trial can proceed with the following trials.

Using BBa_J435300 and the Cas9 plasmid, we perform a transformation. We culture the Escherichia coli in media with IPTG added and media without IPTG added, comparing the colony numbers. (Both media were supplemented with Amp and Cm.) If Cas9 is functioning properly, it can be expected that the colony count in the medium with IPTG added will be less than in the medium without IPTG added. Therefore, if the colony count in the IPTG-added medium decreases compared to the colony count in the medium without IPTG, it can be concluded that the Cas9 has effectively cleaved the AmpR gene (drug resistance gene) in E. coli, and the experiment is successful.


Subsequently, we conducted additional experiments to verify the impact of Cas protein expression on colony formation. We compared the colony numbers of those where Cas protein acting on BBa_J435300 was expressed and those where Cas protein not acting was expressed. The Cas used in the previous experiment is the same as the one used. The non-acting Cas uses a plasmid with the mCherry region unchanged.
We performed a transformation using each Cas plasmid and BBa_J435300, and cultured them in media supplemented with Amp, Cm, and IPTG. This allows Cas protein to be expressed under both conditions. If Cas protein expression does not affect colony formation, it can be expected that colonies will form equally in both media. Therefore, if the number of colonies in the medium with the Cas protein acting on BBa_J435300 decreases compared to the medium with the non-acting Cas protein, it can be concluded that Cas protein expression does not impact colony formation, and the reduction in colony count is due to the action of Cas.