Troubleshooting PCR
Our contribution to other iGEM teams is to share our troubleshooting process with the trials of inverse fusion PCR. Inverse Fusion PCR was attempted many times as a way to add a small DNA fragment and associated linker to a large plasmid. The protocol used was developed by combining information about this process from many protocols from recent research publications. A critical step, however, was initially missed in this development process. The ratio of the insert to the forward and reverse primers should have been 1:500. However, we attempted this PCR process with all three primers in equal concentrations, which will cause the inverse fusion PCR to fail. Additionally, we found that certain polymerases were more effective than others. Because of the small size of the insert and large size of the plasmid vector, only high-fidelity polymerases produced successful replication. For example, in our experimentation, Platinum Taq polymerase was used in several trials, and there was never an instance where DNA was amplified by the PCR enough to be imaged during gel electrophoresis. After transitioning to high-fidelity platinum Taq polymerase, there were consistent PCR amplifications that were extracted from gel electrophoresis. High fidelity polymerases provide a smaller error rate, which is crucial when adding such a small portion of DNA. It is advised to future teams that the extra effort be put forth to invest in a high quality polymerase to produce accurate and consistent results.
Documentation Addition
Our team also added documentation for our encapsulin plasmid as well as the primers we designed for inverse fusion PCR and Gibson assembly as basic parts in the iGEM parts registry. Our encapsulin plasmid part name is BBa_K4037000 and the part is a plasmid that contains the sequence for a T4 T. Maritima encapsulin and mNeonGreen fluorescent cargo co-assembled inside of it. The encapsulin nanocompartment contains HisTag for protein purification as well as SpyTag on the surface of the protein in order to add other molecules to the surface. Alpha factor is expressed at the end of the SpyTag on the surface in order to get the encapsulin system endocytosed into a mammalian yeast cell. Our primers for inverse fusion PCR are parts BBa_K4037001, BBa_K4037002, and BBa_K4037003; the primers for Gibson assembly are parts BBa_K4037004, BBa_K4037005, BBa_K4037006, and BBa_K4037007. Future teams may find this plasmid sequence useful especially if they plan to use encapsulin proteins or wish to add alpha factor to the surface of a protein to trigger endocytosis.