Typically, in order to combine gene fragments, it is neccesary to clone each fragment separately then attach them using Gibson assembly. However, because the secretion tags we wanted to attach to the cargo were small (40-70bp), we first tried to use a technique called site-directed mutagenesis (SDM). SDM allows for the insertion of small gene fragments into double-stranded plasmid DNA via PCR, bypassing the need for Gibson assembly. With SDM, the genetic sequences for the insert is tacked on to the ends of the forward and reverse primers that will be used to copy the backbone. Then, when the primers attach to the backbone during PCR, the insert becomes integrated into the backbone. The end result is numerous copies of the backbone, with the insert added in its proper location.

To construct plasmids via site-directed mutagenesis, we designed and ordered primers that targeted the insertion site and contained the gene for the secretion tag. We used our previously-constructed pcDNA5-sfGFP backbone for these proof-of-concept experiments, exchanging only the secretion tag across the different trials. We ran multiple trials of PCR with the backbone and specific primers, then ran gel electrophoresis to determine if amplification occured. Then we transformed the plasmids into E.coli and let them grow up before isolating the DNA and sending it for sequencing to determine if the secretion tag had been properly inserted.

See "Site-Directed Mutagenesis Protocol" under "Experiments" for further detail.

Because the secretion tags are very short sequences, it is impossible to conclude from gel electrophoresis alone whether or not the tag was properly inserted via PCR. As a result, we had to carry out the entire transformation, culture, and DNA isolation protocol before obtaining sequencing results. Despite several attempts with each secretion tag, upon receiving the sequencing results, only one of the four peptides, HSA, had been properly inserted.

We then successfully transfected the HSA-sfGFP construct into HEK cells and measured secretion of sfGFP, which served as a valuable proof-of-concept (See Results > HSA for further details). However, the overall success rate of site-directed mutagenesis was low for this experiment.

Through our experiments in site-directed mutagenesis, we learned that using small inserts attached to the primers themselves is not always reliable. Colonies were grown, but the plasmids did not contain the insert. Some possible ways to troubleshoot the experiments could be redesigning the primers, increasing the time of the DpnI digest to decrease the likelihood that we transform with an unmodified plasmid, or picking multiple colonies for inoculation into liquid culture to obtain more sets of DNA that could contain the proper insert.

Because we were unable to insert the secretion tags as part of the primers using site-directed mutagenesis, our next idea was to order short fragments containing only the secretion tags. We planned to run PCR on the secretion tags as well as the cargo-containing backbone pieces, then use Gibson Assembly to combine the backbone and the insert. This involves more steps than site-directed mutagenesis, but would possibly be more reliable since we'd already had experience with Gibson Assembly.

We ordered five short DNA fragments, each containing one of the secretion tags (IFNa, IGK8, CTR, SP, and HA). We planned to insert the tags into the pcDNA5-sfGFP backbone we'd constructed, using sfGFP as our test cargo for these experiments. We multiplied the DNA using PCR, ran gel electrophoresis to confirm that we had fragments of the proper sizes, and then attempted to do Gibson Assembly on the fragments. Again, we had to transform the assembled plasmids into E.coli, plate the cells, inoculate into liquid culture, then extract and purify the DNA before we could obtain sequencing results.

After sending the DNA to get sequenced, we found that none of the Gibson Assembly rounds had been effective. Most of the results we had showed the pcDNA5-sfGFP plasmid unmodified, inicating that the small inserts containing the secretion tags had not been inserted.

From these experiments, we learned that Gibson Assembly with very small fragments is difficult. With an insert of only ~60 base pairs, it is likely that the T5 exonuclease enyzyme used for Gibson Assembly digested the fragments before they could be attached. We needed to find a way to attach the secretion tags to the cargo and backbone that didn't involve Gibson Assembly of such small parts.

At this point, we had realized that we could not count on being able to insert or attach the small signal peptides into the larger cargo-backbone constructs using any traditional insertion method. As an alternative, we decided to order longer sequences as gBlocks that contained a signal peptide already attached to one of our cargos. This meant that when we performed Gibson Assembly with the pcDNA5 backbone, we would end up with five completed plasmids, each containing a different cargo with a different secretion tag already attached.

Once we had these plasmids complete, it would theoretically be easier to swap out the cargo sequences and leave the secretion tags attached to the backbones. While the secretion tags are only around 20 amino acids, the cargo molecules are much larger, between 184-1024 amino acids. This would mean that it would be far easier to cut out and duplicate these larger genes, then perform Gibson Assembly to attach them into the secretion tag-backbone constructs.

Constructing the entire catalog of tag-cargo permuations involved many rounds of PCR and Gibson Assembly. Each of the five tag-cargo sequences we ordered had to be inserted into the pcDNA5 backbone. Once this was completed, primers were needed to cut out both the cargo and the tag-backbone fragment from each plasmid. This resulted in a full set of each cargo as well as each tag attached to the backbone. Finally, each cargo could be added to the different tag-backbone fragments. Following Gibson Assembly, all plasmids were transformed into E.coli, for culturing, then the DNA was extracted and sequenced.

With this method of swapping out just the cargo from pre-constructed complete plasmids, we finally saw large-scale success in the construction of our secretion catalog.

Gibson Assembly with the larger cargo fragments was far easier than with the small secretion tag fragments. Despite some challenges with the PCR of the original parts, we were able to successfuly build the plasmids for our secretion catalog.