Team:Western Canada/Results
Preparation of all fragments for assembly
For Gibson assembly as well as TAR-cloning (yeast assembly), good-quality fragments for all backbone parts as well as synthetic inserts needed to be generated with homology between adjacent fragments (Figure 1). To do this, the synthetic DNA parts received from Twist Bioscience were amplified by PCR to have homologous ends compatible the pAGE 2.0 insertion site. In addition, the pAGE 2.0 plasmid was amplified in three overlapping fragments.
Figure 1. Agarose gel electrophoresis of purified PCR amplicons for all fragments. 1 µL of the purified and pooled PCR products for each fragment was run on a 1.4-% agarose gel at 120 V for 40 minutes.
Homology cloning
Although we were unable to yield E. coli transformant colonies from two attempts at the Gibson assembly approach to constructing whole plasmids from the purified fragments, we were successful with our second attempt at yeast assembly (Figure 2, Table 1).
Figure 2. Results from Gibson (top) and yeast (bottom) assembly after transformation of E. coli and S. cerevisiae, respectively. Top panel: DNA from each Gibson assembly reaction was transformed by electroporation (2500 V) into E. coli by mixing 20 µL of reaction product with 200 µL of electrocompetent E. coli EPI300 and spreading 500 µL of recovered cells onto LB 1.5% agar plates supplemented with 30 µg/mL chloramphenicol. Plates were incubated for 24 hours at 37°C. Bottom panel: 20 µL of equimolar fragment mixtures for each construct were transformed into S. cerevisiae spheroplasts using polyethylene glycol (PEG) and 500 the cell-DNA mixture was suspended in each of two tubes containing 8 mL of molten 1M-sorbitol 2%-agar yeast media lacking histidine, poured into empty Petri dishes, and incubated for three days at 30°C.
Table 1. S. cerevisiae colony counts after yeast assembly.
| Plate 1 | Plate 2 | |
| SpyTage:Laccase | 64 | 66 |
| SpyCatcher:CsgA | 93 | 96 |
| CsgA:SpyCatcher | 207 | 151 |
| Laccase:SpyTag | 81 | 78 |
| SpyTag:Cutinase | 107 | 128 |
| Cutinase:SpyTag | 72 | 75 |
Validation of assembled plasmids
Plasmid DNA isolated from the pooled yeast assembly colonies was successfully transformed into E. coli EPI300 (Table 2) for cloning, diagnostic PCR, and restriction digest assays.
Figure 3. E. coli colonies transformed with DNA isolated from pooled yeast colonies. Left panel: 1 µL of DNA isolated from pooled yeast colonies was transformed by electroporation (2500 V) into 40 µL of electrocompetent E. coli EPI300. 700 µL of recovered cells were spread onto LB plates supplemented with 30 µg/mL chloramphenicol and incubated for 24 hours at 30°C. Right panel: 20 colonies from each transformation were selected and streaked onto fresh LB plates supplemented with 30 µg/mL chloramphenicol and incubated for 24 hours at 30°C.
Table 2. E. coli colony counts after electroporation.
| Count | |
| SpyTage:Laccase | 123 |
| SpyCatcher:CsgA | 136 |
| CsgA:SpyCatcher | 135 |
| Laccase:SpyTag | 534 |
| SpyTag:Cutinase | 117 |
| Cutinase:SpyTag | 83 |
To identify successfully-assembled plasmid clones, we performed colony PCR using lysate from individual E. coli colonies for each construct. The pAGE 2.0 vector has a pair of primers flanking the insertion site, which is the junction between fragments C and A (Figure 4). Performing PCR with these primers yields an amplicon sized either 214 bp if no insertion is present or 214 + insertion size if there is an insertion present (Figure 5). For reference, pAGE 2.0 with no insert was used as template DNA for PCR using these primers (+).
Figure 4. Annotation map of the insertion site in pAGE 2.0. Figure was generated using Geneious version 2019.2, created by Biomatters (Available from https://www.geneious.com).
Figure 5. Gel electrophoresis of colony PCR products from lysed E. coli cells from colonies containing assembled plasmids. 1 µL of the products from the colony PCR of the plasmid insertion site (Figure 4) was run on a 1.4-% agarose gel at 120 V for 40 minutes. PCR products yielding electrophoresis bands of the right size are identified with green circles.
From these colony PCR results, we selected putatively-positive clones (circled in green) and inoculated cells from the corresponding colonies into selective liquid media to grow overnight. In the morning, 1 mL from each saturated culture was transferred to a 250-mL culture flask containing 50 mL of selective liquid media supplemented with arabinose to induce expression through the pBAD promoter. Our constructs were designed such that pBAD drives an operon encoding the inserted open reading frame as well as monomeric red fluorescent protein (mRFP), both of which are preceded by a ribosome binding site (RBS). The diluted & induced cultures were placed for six hours at 37°C in an incubator set to shake at 225 rpm.
The cultures were transferred to 50-mL Falcon tubes and centrifuged at 3000 rcf for 25 minutes at 4°C. The supernatants were discarded and the pelleted cells were observed for the presence of red fluorescence (Figure 6). Since our inserts are polycistronic with, and upstream of, mRFP, red fluorescence indicate that our inserts are at least being transcribed. Since the same RBS sequence was used for both open reading frames, we can also reasonably predict that fluorescing cells are producing our synthetic proteins.
Figure 6. Pelleted cells from induced cultures of E. coli containing putatively-positive clones of each plasmid. Cultures were centrifuged for 25 minutes at 3000 rcf at 4°C after inducing for 6 hours with 98 µg/mL arabinose and 225-rpm shaking at 37°C.
Based on the presence of red fluorescence, we predict that the following clones are likely expressing our synthetic proteins:
- SpyCatcher:CsgA (A) – 1, 2, 4, and 5
- CsgA:SpyCatcher (B) – 1, 6, and 7
- SpyTag:Laccase (C) – no fluorescent pellets observed, which was to be expected due to unsuccessful colony PCR
- Laccase:SpyTag (D) – 8, 11, and 14
- SpyTag:Cutinase (E) – 4 & 5
- Cutinase:SpyTag (F) – 1, 2, 3, 4, and 5
DNA was then isolated from all pelleted cells using the EZ-10 Spin Column Plasmid Purification Kit from BioBasic Inc. With isolated plasmid DNA, we then repeated the same diagnostic PCR protocol (Figure 7).
Figure 7. Gel electrophoresis of PCR products from isolated plasmid DNA from induced E. coli cultures. 1 µL of the products from the diagnostic PCR of the plasmid insertion site (Figure 4) of each assembled plasmid was run on a 1.4-% agarose gel at 120 V for 40 minutes. PCR products yielding electrophoresis bands of the right size are identified with green circles.
These PCR results further confirm the fluorescence observations, with red pellets corresponding with a PCR amplicon band of the correct size for gene insertion and plasmid assembly. The exception to this is clone 1 of the SpyTag:Cutinase construct, which yielded a correctly-sized band without visible fluorescence in the pelleted cells. This discrepancy suggests that while the gene SpyTag:Cutinase gene was successfully inserted into the plasmid, there may have been spontaneous mutations to the pBAD promoter region or the mRFP cistron/RBS.
To further probe each assembled construct for successful assembly and overall integrity, we digested samples of each plasmid with the high-fidelity restriction endonuclease EcoRV-HF from New England Biolabs. For each plasmid, this restriction enzyme should yield a banding pattern that differs from that of pAGE 2.0 without any inserted fragment (Figure 8).
Figure 8. Gel electrophoresis of EcoRV-HF restriction endonuclease digest products from isolated plasmid DNA from induced E. coli cultures. Top panel: expected banding pattern from an EcoRV digest of each assembled plasmid. This figure was generated using Benchling’s virtual digest feature. Middle and bottom panels: 300 ng of each isolated plasmid was digested with 10 units of EcoRV-HF restriction enzyme in a 20-µL solution containing 2 µL of CutSmart. After incubating for one hour at 37°C followed by 20 minutes at 65°C, the entire solution was run on a 1.4-% agarose gel at 120 V for 40 minutes. Lanes with the expected banding pattern are identified with green circles.
The restriction digest results largely confirm the PCR and fluorescence results described above, however there are some unexpected banding patterns for some clones. Surprisingly, the banding patterns for clones 8 and 11 of the Laccase:SpyTag construct are closer to the pattern expected for SpyTag:Laccase. As with the PCR result for SpyTag:Cutinase clone 1, the banding pattern suggests a properly structured plasmid, which contrasts with the absence of fluorescence. These unusual findings require further assays, such as sequencing, to elucidate the underlying plasmid structure.
Sequencing of insertion regions
The PCR reaction products for each fragment were submitted for Sanger sequencing at the Robarts Research Institute DNA Sequencing Facility using the same primers as for PCR. The sequence data we received was analyzed by mapping each read to the corresponding reference sequence and disagreements from the reference sequence were highlighted (Figure 9). The raw sequence results are available at the end of the lab Notebook.
Figure 9. Consensus sequences from bidirectional Sanger sequencing of the insertion sites of each assembled plasmid. Forward and reverse Sanger sequencing results of the PCR amplicon of the insertion site of each assembled plasmid were aligned to their corresponding reference sequences. The consensus of each pair of sequencing reads was annotated, regions which aligned with the reference were shaded in grey, and disagreements (including ambiguous base calls and no read coverage) were highlighted in black. Figure was generated using Geneious version 2019.2 by Biomatters (available from www.geneious.com).
This is how far our team has gotten with our iGEM project. Next year, Western Canada’s iGEM team will continue where we left of by transferring clones with correct sequences to expression strains and performing functional experiments to characterize each part.