PCR of Gene-of-Interest

We made more of the human alpha-amylase gene insert (obtained from Integrated DNA Technologies) through PCR amplification. The human alpha-amylase gene insert (GOI) appeared to be in the right location–between 1000bp-1500bp in length. Between 1kb and 2kb of the PCR products ran clean because of the water.

plasmid backbone
Figure 1: 07-25-23 PCR of GOI:

Generation of Plasmid Backbone

We saw that the plasmid aligns with the 5kb mark of the DNA ladder. All six wells were 5kb in length, which was the result expected based on the sequence length of the plasmid backbone. However there was no clear indication that the 650bp product of the excised sequence of the plasmid backbone was present. Although there are faint DNA bands along the 500 bp mark of the DNA ladder, they are most likely ghost bands“: artefactual bands frequently occurring, especially when partially homologous nucleic acids, such as splicing variants of DNA transcripts, are analyzed simultaneously.”

plasmid backbone
Figure 2: 07-28-23 Restriction Digest of plasmid for 60 min at 42C

The results for the gel electrophoresis of the restriction digest of our PET-28a plasmid appear at 5kb at 37C for 15 min and 60 min. We chose to test whether time was an issue because the gel results from our previous restriction digests were incoherent with the expected placement of the DNA bands in the DNA ladder. The protocol that we followed recommended we leave the restriction enzyme + plasmid backbone assay for 15 minutes. We hypothesized that maybe we didn’t give the restriction enzymes enough time to excise the DNA. Our hypothesis was proven wrong and based on these results, Time is NOT the issue; the plasmid moves the same length regardless of length of time kept at 37ºC. The 650 bp product is still not in the gel electrophoresis, making it clear to us that our first attempt at restriction digest was not a fluke or an anomaly.

plasmid backbone
Figure 3: 08-02-23 Restriction digest of plasmid for 15 min or 60 min at 37C

We tried increasing the concentration of the plasmid backbone in the assay with the two restriction enzymes to see whether there was not enough of the plasmid for the restriction enzymes to efficiently cut. The DNA bands for the plasmid appear at 5kb at 37C for both 15min and 60min, further confirming our assumptions that time didn’t affect the restriction enzymes’ ability to cut the plasmid backbone. We also learned that concentration is NOT the issue because the DNA bands for the plasmid move the same length regardless of the concentration of restriction digest of the plasmid. Both decreased/increased combinations of time and decreased/increased concentrations of plasmid backbone in the assay containing the backbone and restriction enzymes did not seem to result in the 650 bp product.

plasmid backbone
Figure 4: 08-09-23 Restriction digest of plasmid for 15min OR 60min at 37C with DNA gradient

We tried different combinations of the restriction enzymes with the plasmid backbone to test for three possible reasons for the MCS not being properly excised: the XbaI restriction enzyme wasn’t working, the XhoI restriction enzyme wasn’t working, or both of the restriction enzymes weren’t working. Just like the other gels, the Plasmid appears at 5kb at 37C when run through a thermocycler for 15 minutes and for 60 minutes. The DNA bands for the assays with only one of either restriction enzymes clearly indicated that the enzymes were cutting the backbone as shown in the difference between the length of the uncut plasmid backbone and the individually cut plasmid backbones, but we didn’t see any 650 kb product. We had several new hypotheses after this result: Enzymes are nicking the plasmid BUT not cutting the plasmid? Are the restriction sites mutated, not allowing the enzymes to cut? We sent out our plasmid for sequencing to help answer these questions.

plasmid backbone
Figure 5: 08-09-23 Restriction digest of plasmid for 30min at 37C with 1μg DNA

We ran a PCR on the multiple clonal site to determine if that was the cause of our error. The MCS appears to be around 0.7 kb long, which is close to the expected length of our excised sequence (0.65 kb). We came up with possible reasons for why the restriction enzymes weren’t working after determining that the PCR of the flanking regions of the MCS were in fact around 0.65 kb long. There was a chance that we might have run the gel for too long. There was also a possibility that there was not enough of the plasmid backbone for the restriction enzymes to sufficiently locate and cut the target sequence. The volume of samples inserted into the well could have also been insufficient for the DNA to run completely in the gel.

plasmid backbone
Figure 6: 08-11-2023 PCR to the flanking regions of the MCS (Multiple Cloning Site)

At one point, we decided to start from scratch and isolate the PET plasmid DNA using a completely new mini-prep kit, as we realized the possibility our mini-prep kit was malfunctioning. We measured 44.483 ng of plasmid DNA per uL * 50 uL of plasmid DNA solution * 6 samples ~ 13.2 ug of DNA. This isolation of PET plasmid DNA using the new kit led to more of the plasmid being isolated compared to when the previous QIAgen miniprep kit. We decided to use this plasmid for mini-prep going forward.

plasmid backbone
Figure 7: 09-04-23, Isolation of PET plasmid DNA using new mini prep kit

Although we reordered a new plasmid (to see if the plasmid itself was the problem), the results showed that the restriction enzymes were still not cutting plasmid. This led us to consider future steps for us to consider in resolving this issue. We thought about ordering new enzymes because the restriction enzymes themselves might have been inaccurately chosen for our specific plasmid. We also considered the possibility that the plasmid is not what we think it is. In other words, either the design of the plasmid on benchling was inaccurate, leading to further errors in choosing the suitable restriction enzymes.

plasmid backbone
Figure 8: 09-07-23, Restriction digest of NEW plasmid for 30min at 37C with 1ug DNA following results from 08-11-23

We tested the new restriction enzymes that we ordered on the new overnight culture, changing two potential variables that could have explained the failure of our previous trials. We also used the highest resolution gel to date. The results still showed missing excised MCS, meaning none of the variables/conditions listed above were issues. A possible pivot we considered was to move to utilizing either a western approach (technique used to separate a mixture of proteins based on weight using gel electrophoresis) or an aptamer approach, to begin testing the viability of aptamer-based detection. We started to develop a new research plan for October to move past our restriction digest challenges to start aptamer testing…

plasmid backbone
Figure 9: 09.26.2023, Restriction digest with new enzymes

Sequencing Using Plasmidsaurus

After receiving these results, we felt that we had no other option but to ship in our samples for sequencing to see if our problem was due to an error in the sequence itself. We attempted to send our samples to Plasmidsaurus. However, the company notified us that they were not able to obtain any results, likely do to nuclease degradation.

Figure 10: An email from Plasmidsaurus explaining that our plasmid sequencing did not work.