Reporter module and its controls (GFP + RFP)

We were able to clone parts of the full GFP + RFP reporter, as shown by both the successful colony PCRs, digests, and sequencing results. However, due to the cloning issues with the CUG promotor and unexpected recombination when an alternative inducible promoter was added, we were unable to create a fully functioning reporter plasmid. Our results are illustrated and further discussed below.

Figure 1: A colony PCR showing the successful cloning of pSB1C3_BBa_K4720024 on a 2.0% agarose gel. The samples in the green boxes signify the successful fragment size.

As can be seen in Figure 1, the colony PCR showed several successfully cloned pSB1C3_BBa_K4720024 constructs. The construct is the result of the RFC[10] standard assembly with BBa_R0051 (Pcl)  as an insert and pSB1C3_BBa_K516030 (RBS+RFP+TT) as the backbone. We chose a 2% Agarose gel instead of the standard 0.9% gel. This is because the difference between the correct and incorrect fragment size is only 57 bases (see R control). The correct fragment size is 1235 bp. We sent R2, R5, R6, and R7 for sequencing to confirm our results. R6 and R7 were confirmed to be the desired construct by Sanger sequencing. 

Figure 2: A successful gel electrophoresis of a digest of the previously shown C7 (figure 2: R7) pSB1C3_BBa_K4720024 (backbone) and the digested PCR product of the BBa_K4720013 (GFP) (insert). 

The PCR procedure was necessary because both fragments were on a chloramphenicol resistance marker plasmid. The PCR product was digested with DpnI to remove the template plasmid. The fragment sizes as seen in Figure 2 are correct. The Linear GFP sample had to have a fragment size of 898 bp, and the RFP+C7 construct had an expected size of 2976 bp. These digests were used for the ligation of pSB1C3_BBa_K4720015. 

Figure 3: Gel depicting NotI digest of pSB1C3_BBa_K4720015 plasmid, showing incomplete digestion. Sanger sequencing confirms the correct construct in both plasmids.

The gel in Figure 3 shows the restriction digest of the suspected pSB1C3_BBa_K4720015 plasmid. The plasmids were digested with NotI. This should result in two fragments of 1828 bp and 2046 bp. The template concentration must have been too high for the amount of enzyme used, as we see an incomplete digest. The digest result however can be seen for samples 2.1 and 2.2. 1.3 also shows these fragments, but shows other contamination besides that. The results from the Sanger sequencing show that both plasmids contain the right construct.

Figure 4: Sequencing results for GFP + RFP construct (plasmid pSB1C3_BBa_K4720016) showing recombination at the BBa_B0015 terminator sequence (indicated by the red bar at the bottom). Alignment was performed using the MAFT7 algorithm. Image source: Benchling.

To test the GFP + RFP construct, the Ptac promoter was put in front of GFP resulting in plasmid pSB1C3_BBa_K4720016. This plasmid was however not obtained, because recombination took place on the repeating double terminator sequence BBa_B0015. In Figure 4, the sequencing result is aligned with a section of the desired template. The red bar at the bottom indicates the missing section. Image obtained from Benchling. Alignment algorithm MAFT7: https://academic.oup.com/mbe/article/30/4/772/1073398

Figure 5: Colony PCR results for plasmid pSB1C3_BBa_K4720012, designed to eliminate repeating sequences (GFP - BBa_E0040 + new terminator - BBa_B1006).

To remove repeating sequences, pSB1C3_BBa_K470012 was designed (GFP (BBa_E0040) + new terminator (BBa_B1006)). Figure 5 gel shows the result of a colony PCR for plasmid pSB1C3_BBa_K4720012. The expected fragment size is 1079 bp. This is observed in samples 1, 2, 3, 6 and 7.

Hydrolase

To prevent E. coli-produced cyclic di-GMP from influencing our results, we cloned a plasmid of cyclic di-GMP hydrolase to reduce endogenous cyclic di-GMP concentrations.

Figure 7: Colony PCR results of the pSB3T5_yhjH construct. Samples 7 and 12 (green box) had promising construct sizes and were sent for sequencing. 

We sent Samples 7 and 12 for sequencing to confirm our result. Both were confirmed to be the desired construct by Sanger sequencing. We also successfully transformed pSB3T5_yhjH into our endpoint host, E.coli TG1.

Sensor module (PpeI + FleQ)

We were able to clone parts of the full GFP + RFP reporter, as shown by both the successful colony PCRs, digests, and sequencing results. However, due to the cloning issues with the CUG promotor and unexpected recombination when an alternative inducible promoter was added, we were unable to create a fully functioning reporter plasmid. Our results are illustrated and further discussed below.

Figure 8: Gel analysis confirms successful Phusion PCR amplification of essential Gibson Assembly fragments: pSB1A3 backbone, FleQ gene, and promoter.

The gel analysis revealed the successful amplification of the fragments crucial for the Gibson Assembly process through Phusion PCR. These fragments include the pSB1A3 backbone with an expected size of 2155 bp, the FleQ gene fragment spanning 1578 bp, and the promoter fragment measuring 429 bp. The attainment of these expected fragment sizes demonstrates the effectiveness of the PCR amplification, indicating that the essential components for the subsequent Gibson Assembly process were correctly generated. 

Figure 9: : The colony PCR for the CUG promotor after the 1st attempt at Gibson assembly (unsuccessful). This result already hinted at what the sequencing result confirmed: complementary sequences in the plasmid were binding to each other and creating constructs of various sizes.

Figure 9 shows the colony PCR of the suspected cyclic-di-GMP sensor plasmid. We expected a fragment size of 2231 bp, but unfortunately, none of the 16 samples had the correct fragment size. Notably, samples 2, 6, 7, 8, and 11 showed multiple bands. This, in combination with the large variety of fragment sizes, suggests that there are some complementary sequences within the plasmid that are binding during the Gibson Assembly, resulting in these varied fragments. We sent samples for sequencing, where it was confirmed that the construct was not successfully abstained and that it was likely the repeating sequence of the Pcl promoter that was causing recombination and subsequent loss of the FleQ coding sequence. This is why we changed the approach to Golden Gate with SapI, as this assembly method has specific sticky ends. 

Figure 10: Gel depicting PvuI Restriction Digest of c-di-GMP sensor module-derived plasmids from Golden Gate assembly with SapI

In this experiment, a PvuI restriction digest was performed on linearized plasmids derived from the c-di-GMP sensor module through Golden Gate assembly with SapI. The expected fragment size was 4072 bp, while the control fragment measured 3224 bp. All resulting plasmid types underwent sequencing for further analysis. Sample 4 represented an empty pSB1A3 vector, while sample 5 contained the Pcl+Ppel insert (BBa_K4720018) and served as a recombination control. Sample 6 involved ligation with only the promoter insert, and sample 8 represented a small empty vector. 

Figure 11: Gel depicting the restriction digest of pSB1K3_BBa_K4720017 using EcoRI and BcuI for subsequent combination with BBa_K4720012 into pSB1C3_BBa_K4720021. Unexpectedly, fragment sizes did not align with the anticipated 1926 bp and 2181 bp sizes. Lanes 5.3, 5.4, and 5.7 display two bands near the expected range, alongside additional bands. 

The experiment involved a restriction digest of pSB1K3_BBa_K4720017 using EcoRI and BcuI as part of the plan to combine it with BBa_K4720012 into pSB1C3_BBa_K4720021. Unfortunately, the expected outcome did not materialize due to mismatched fragment sizes. The anticipated fragment sizes were 1926 bp and 2181 bp, but in lanes 5.3, 5.4, and 5.7, two bands around this expected size range were observed, along with the presence of additional bands. It's worth noting that we unintentionally sent the wrong samples (5.1 and 5.2), which contained RFP for unknown reasons, for sequencing. We are currently awaiting sequencing results to gain further insights into this unexpected outcome.

Biosensor control modules (Construct 1+3B, control for FleQ)

Figure 12: Gel analysis of NotI restriction digest of pSB1C3_BBa_K4720020. Unexpectedly, no expected fragments were found. Samples 3.1 and 3.6, sequenced afterward, revealed issues with the Pcl+Ppel tandem promoter in 3.1 and contamination in 3.6. The observed fragment size (3067 bp) suggests potential incomplete digestion, requiring further investigation.

The restriction digest of pSB1C3_BBa_K4720020 with NotI aimed to yield fragment sizes of 1021 bp and 2046 bp, as expected. However, the observed results did not align with this anticipation, with no matching fragments detected. Notably, two distinct types, labeled 3.1 and 3.6, were visible on the gel, and both were subsequently sent for sequencing. Sample 3.1 was found to be missing the Pcl+Ppel tandem promoter, while sample 3.6 contained the correct construct but displayed contamination issues, necessitating purification. Interestingly, if the digest were incomplete, with only one cut, the expected fragment size should be 3067 bp, which corresponds to the observed fragment.

Figure 13: Sequencing of sample 3.6, aligned with pSB1C3_BBa_K4720020, showed misalignment beyond GFP coding. The chromatogram revealed two sequences: GFP coding and suffix with pSB1C3 backbone. Suggests that pSB1C3_BBa_K4720020 may be obtained with mixed sequences. Resolution: dilution streaking and screening for correct plasmid isolation.

The sequencing results for 3.6 yielded an intriguing observation when aligned with the target sequence of pSB1C3_BBa_K4720020. It became evident that the fragment did not align entirely beyond the start of the GFP coding sequence. A closer examination of the chromatogram revealed the presence of two distinct sequences. One of these sequences corresponded to the GFP coding sequence, while the other encompassed the suffix and pSB1C3 backbone. To resolve this discrepancy, we examined the peaks that did not align with GFP and then matched this sequence with the pSB1C3_BBa_K4720020 plasmid map. These findings suggest that the pSB1C3_BBa_K4720020 plasmid may have been obtained, albeit with an issue of mixed sequences. To address this, a potential solution would involve dilution streaking of the colony material followed by a thorough screening of the new colonies to isolate the correct plasmid.