Team:SIAT-SCIE/Protocol

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PCR

Primestar Max PCR protocol — for plasmid

1. General Composition of PCR Reaction Mixture

NOTE: When using plasmids as the template, the mass of the mixture should not exceed 1ng.

2. PCR condition

Before cycles: 98°C 10 After cycles: 72°C 7min; 16°C ∞ (4°C is more suitable for storing PCR product, but storing 4°C overnight may result in damage of some PCR equipments)

Mechanism of FadA protein

The FadA protein is activated when its two forms combine and become internalized. The first form is a pre-FadA that is anchored in the cell membrane, whereas the second form is the mature FadA (mFadA) that is secreted out of F. nucleatum. When the two forms combine to form a complex, the protein is capable to help F. nucleatum bind to the host epithelial cell, thus allowing F. nucleatum embark on invading the host cells.

Overview (Fig. 1)

We will first express Cas9 and sgRNA in E.coli and then transport them to E.coli’s periplasm(Step 1). By then, they may be packaged by OMVs that bud off from E.coli’s outer membrane(Step 2). Those OMVs will be collected and mixed with our target bacteria, thereby allowing them to fuse with bacteria again, to release the Cas9 proteins and sgRNA(Step 3), and cleave the target gene (Step 4 & 5). Afterwards, we will test whether the target gene is cleaved by Cas9.

Safety

Out of safety concerns, instead of using the pathogenic Fusobacterium nucleatum, we transform a section of FadA’s coding sequence — with sgRNA’s binding site — into E.coli to test our system’s efficiency.