Introduction
This year, our contributions to future iGEM teams focused on creating new parts in the parts registry and documenting our troubleshooting processes. We hope that future iGEM teams will be able to use our parts and documented experiences to further advance their team’s research.
Documentation of Encapsulin, AMP, and Targeting Peptide
Our team added documentation for our antimicrobial peptide (AMP), T4GALA encapsulin, TEV Protease site, and targeting peptide as basic parts in the iGEM parts registry. We also registered the AMP + T4GALA + targeting peptide assembly as a composite part in the registry. Our AMP part name is BBa_K4143336 and this part contains the sequence for HBCM2, an α-helical hybrid of cecropin and melittin. To make this AMP, we codon-optimized the sequence found in literature to be compatible with the BL21 strain of E. coli that we used for expression. Our encapsulin part name is BBa K4143337 and this part contains the sequence for the T4GALA encapsulin. Our encapsulin was chosen because lowering the pH of the environment to 6 triggers disassembly of the encapsulin nanocompartment. This allows the AMP + targeting peptide fusion to be released from the encapsulin and subsequent cleavage by TEV protease. Our targeting peptide part name is BBa_K4143339. This part serves to direct cargo for sequestration inside an encapsulin. Finally, the composite AMP + targeting peptide + encapsulin part name is BBa_K4143340. Future teams would find this composite part useful if they desired to use our expression system for production of a toxic protein/peptide biosynthetically in E. coli.
Troubleshooting
Sequencing pETDuet Plasmids
One contribution to other iGEM teams is our troubleshooting process with plasmid sequencing results. After Gibson assembly, we chose generic primers that overlap with the T7 promoter from the sequencing department at our university and sent the plasmid for sequencing to check whether the AMP construct inserted correctly. Initially, our sequencing results were poor in the region where we expected to see our AMP sequence. Interestingly, we noticed that our sequencing was bad for about 230 base pairs, with many bases being marked as “N” for unknown. A DNA polymerase usually drops off early if there are poor sequencing results but ours went to about 1100 bases, which is a good read length. We did notice, however, that our target peptide and linker sequences, which are unique sequences towards the end of our sequence reading, did appear to be inserted correctly. The presence of these irregular sequences made us think that our transformation may not have been completely unsuccessful and that there may be an issue with our sequencing method.
After analysis, we realized that there are two T7 promoters in the pETDuet plasmid we were using. When our plasmid was sequenced, DNA elongation began at both T7 promoters, meaning sequencing was occurring at two locations on the plasmid instead of one. These strands started from different places and therefore were not identical, so the sequencing returned convoluted data. To solve this problem, we made custom primers for sequencing that corresponded to a unique site upstream of our insert. Using custom primers resulted in successful sequencing. It is advised that future teams make sure to check primers for multiple T7 promoters before using premade T7 primers for sequencing, particularly if using a pETDuet plasmid.
DNA Gel Electrophoresis
Our team can also share our troubleshooting process with smearing gel results from gel electrophoresis. When we began lab work, our gels repeatedly showed smeared, unclear bands when imaged. We initially ran gels with control DNA to test whether our DNA was causing the problem. The gels with control DNA looked just as smeared as the gels with our experimental DNA, so we knew that our samples were not causing an issue. Next, we experimented with different parameters to improve our gels. We tried running gels at a lower voltage, a longer time, and measuring the temperature of the gels more precisely before they were poured (using a digital instead of an analog thermometer). These improvements made our gels slightly clearer, but increasing the agarose percentage of our gels from 0.5% to 1% resulted in the most notable improvement in clarity of bands on the gel. The higher the concentration of agarose within the gel created smaller pores within the matrix. Smaller pores make it difficult for large molecules to migrate through the gel and so they favor the separation of fragments in the sample. We advised that future iGEM teams match the agarose concentration of their gels to the size of DNA fragments they expect to see before they start running gels.