The protocols describes in detail the ideas and experimental procedures of our project, and the purpose of each experiment and related materials are also listed. We provide detailed information for the iGEM team member who wants to learn more about our project. We also hope to have in-depth exchanges with other teams.
The plasmids of His-SUMO-Cas 13b-Psm and His-SUMO-Cas 13b-Cca plasmids were constructed by one-step cloning. In this paper, we introduce our method with the example of PsmCas 13b plasmid. Firstly, we linearized the plasmid vector carrying the PsmCas 13b gene, and introduced the end sequence of the linearized vector at the 5 'end of the PCR primer of the inserted His-SUMO fragment, so that the 5' and 3 'ends of the PCR fragment had the same sequence as the two ends of the linearized vector respectively. Then, the amplified SUMO-His fragment was mixed with the linearized Psm vector in a certain proportion, and the recombinant enzyme was used to catalyze the transformation at 37 ℃ for 30 min to complete the directional cloning.
For detailed experiments of each step, please refer to Cloning protocol.
The plasmid with virus sequence was transferred to the competent E. coli to simulate the real detection samples, and the samples were subjected to experimental operations such as plasmid linearization, transcription, RT-RPA, etc. , so that the target sequence could reach the concentration detected by Cas13 protein. We used RT-RPA technology to make sample processing easier in the device, while the previous transcription of RT-RPA was to collect more data for modeling.
For detailed experiments of each step, please refer to sample processing protocols.
We expressed our Cas13 protein in E. coli strains. The proteins have 6×His tags, which allows us to use Ni-NTA Agarose Resin for purification. There are "Äkta Start" protein purifiers in our laboratory, so we prefer to put the Ni-NTA Agarose filler into a pre-packed column for purification with a protein purifier. But the purification results are not ideal. After that, we tried the gravity method, which can incubate the protein with Ni-NTA Agarose Resin for one hour so that the protein can fully bind with Ni-NTA Agarose Resin. The latter has better purification effect. SUMO and MBP tags can increase the solubility of proteins and prevent them from forming inclusion bodies. But too large a tag will affect protein activity, so we add SUMO protease or rTEV protease to cut His-SUMO or His-MBP tags during dialysis. After digestion, the protein can be separated from the label by purifying it again. The purification result can be viewed by SDS-PAGE.
For detailed experiments of each step, please refer to Cas 13a&b protocols.
Cas 13 protein informations:
As the final application of our product, we lyophilized the detecting system to extend the shelf life of our products. But lyophilization may have effects on the activity of Cas13. So we designed experiments to test the effect. Besides, we made lyophilized chip to test the performance of our device.
For detailed experiments of each step, please refer to Lyophilization protocols.
To make our detecting result visible,we chose fluorescence as our read out format.We designed fluorescence reporter(a fluorophor in the one end and a quencher in the other,the linking oligonucleotide are corresponding to specific Cas13 protein) which is based on the fluorescence resonanceenergy transfer (FRET) to achieve our goal.When the target exists in the system,the collateral cleavage of Cas13 protein will be activated,then the reporter will be cut off, and be turned on,then the fluoreacence emits. We tested the cleavage activity, the detecting limitation,and the cleavage base preference of Cas13 protein orthologs.
For detailed experiments of each step, please refer to Readout protocols.
Our fluorescence detector can draw dynamic curves of the changes of four fluoresceins in a sample at the same time, and finally store them in an SD card. It is equivalent to a small microplate reader with high sensitivity, but because the sample carrier is a filter paper, it cannot reach the precision level of the microplate reader. This device is relatively complicated to use, and the method of use is written in the relevant instructions.
For detailed experiments of each step, please refer to Device test protocols.