Our Process

Our experimental design has been built upon many previous generations of senior design students, as well as the scientists at JCVI. The main steps include designing components, mass producing and assembling these components using E.coli cloning, and finally transforming into a mycoplasma cell for growth.

The first design step focuses on the synthesis of both the guide RNA for CRISPR and the barcode for strain identification. The barcode is made by having an 8 base pair region in the plasmid which randomly generates a barcode during cloning. The gRNA were previously synthesized and designed to match the desired gene to knock out.

Next, we inserted the gRNA into our backbone while generating the barcode. E.coli transformation was used because process can take up foreign DNA, assemble it into its own DNA, and can mass produce it. Not only does this allow us to assemble our plasmid, but it also makes many copies of this plasmid for later use. After the E. coli cells grow, we then extracted the DNA from the cell through a process called mini-prepping. This process breaks down the E.coli cell, removes all cell membrane parts, and results in purified DNA. During this step, we run an agarose gel to ensure that the DNA is the correct size and help confirm the validity of the sample. If this checks out, then the DNA is ready to be transformed.

The final stage is the most vital stage as it includes transforming our engineered plasma into a mycoplasma cell. After performing a mycoplasma transformation, cells must be plated for 4-5 days to allow the mycoplasmas to grow. Single colonies are then picked and further grown. It is very important to get a single colony during this period to ensure we are getting pure strains with unique barcodes. To check our final product, we amplify the DNA in our sequences through junction PCR and then analyze through Sanger sequencing. Sequencing results illustrating base calling through an electropherogram. The top figure exemplifies clear, ideal base calling while the bottom figure depicts noise. The bottom, noisy base calling could result in inaccurate sequencing results that could make us believe we have mutations where there otherwise is not. Thus, samples that have unclear reads must be resequenced to ensure accuracy.

This confirms that our engineered plasmid worked and is free of unwanted mutations. For assurance, we picked three colonies for each to ensure we get at least two good samples, a primary and a backup. If ineffective, other colonies must be picked and sent for sequencing. If sequences are correct, sample is frozen down and added to library.

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