RESULTS | UCSC - iGEM 2023

Summary of Results

Our project was primarily successful in its bioinformatic and sequencing aspects. We have successfully sequenced the bidirectional promoter regulating the genes responsible for microcystin synthesis in UTEX 2385 M.aeruginosa, enabling specific targeting of the mcy cluster to halt microcystin toxin production in the future. We have also successfully prepared a partial assembly of the UTEX 2385 M. aeruginosa genome, validating it using Cluster-K (see Engineering). Our Chameleon software project has been completed and validated via its ability to remove restriction sites on DNA sequences in silico. A consensus RBS for UTEX 2385 M. aeruginosa has been identified and strength-assessed using bioinformatic techniques and made available on the iGEM Parts Registry. Fragments for Golden Gate assembly of the modified and unmodified pSPDY plasmids were prepared via amplification of gBlock Gene Fragments with flagged primers and verified.

Sequencing mcy gene cluster promoter region

In order to silence expression of downstream microcystin synthetase genes in our future integrative plasmid, we needed to sequence the bidirectional promoter of the mcy cluster in UTEX 2385 M.aeruginosa [1]. Partial CDSs of the synthetase genes and parts of the promoter region have previously been sequenced in UTEX 2385 and other M. aeruginosa strains, however the region spanning from mcyD to mcyA has not been previously sequenced in this strain. Since most of the region was unsequenced before our work, designing primers to generate an amplicon covering the region presented a challenge; by using regions of the mcyD and mcyA partial CDSs from other strains, we were able to design two primers that would bind to the genes flanking the promoter region. Using these primers in a PCR reaction with UTEX 2385 M. aeruginosa genomic DNA as template, an amplicon of the promoter region was created. The amplicon and primers were then sent out to the UC Berkeley DNA Sequencing Facility for Sanger sequencing; the resulting chromatogram was analyzed to produce the nucleotide sequence of the mcy cluster promoter region.

Figure 1. Annotated consensus sequence of a 1,131 BP region covering part of the mcyD gene, the bidirectional promoter region, and part of the mcyA gene of *M.aeruginosa* UTEX 2385. Annotations were made based on alignment to sequences in the published *M.aeruginosa* PCC 7806 mcy cluster. Image rendered in Geneious Prime.

Partial sequencing and Cluster-K validation of M. aeruginosa genome

Figure 2. Flye assembly visualized by Bandage: Colored contigs indicate correlation to the *M.aeruginosa*'s genomic DNA.

To implement the Stealth technique on M.aeruginosa and create a plasmid modified by the Chameleon project, we initiated the process by sequencing the genome of the UTEX 2385 M. aeruginosa strain using the MinION platform from ONT. Subsequently, a substantial amount of genomic DNA, approximately 4 out of 6 megabases, was successfully assembled through Flye and visualized with Bandage, as depicted in Figure 2.

The assembled data underwent further processing to extract the 16S rRNA sequences. These sequences were then subjected to a Nucleotide BLAST analysis, which revealed that only three contigs (namely contig 17, 2, and 8) were confirmed to belong to M. aeruginosa. Given the complex nature of our culture, it is important to note that M.aeruginosa exists as a xenic culture, meaning it coexists with various other bacterial species. To ensure the accuracy of our findings, an additional verification step was deemed necessary.

To address this, we employed Cluster-K, a tool that not only supported the 16S rRNA data but also introduced potential candidate contigs that might be associated with M.aeruginosa (Figure 3, 4). It is worth emphasizing that Cluster K's utility extends beyond our specific case, particularly in metagenomic analyses and environmental samples where the diversity of species to be analyzed is considerably higher.

For more details on Cluster-K see the Engineering Page cycle 3

Figure 3 Cluster-K Output: Cluster 1 (in orange) predicts that elements of the cluster belong to the same species,*M. aeruginosa*

Figure 4. A Venn diagram of BLASTN and Cluster-K (group 1) output

Design of Synthetic M. aeruginosa RBS

Using the Salis RBS Library Calculator v2.1.1, a synthetic RBS with a high predicted translation initiation rate (TIR) for M.aeruginosa was generated [2], [3], [4]. Generating the RBS required a CDS sequence and specified organism; the sequence of our eGFP gene optimized for expression in UTEX 2385 M. aeruginosa(documented on the BBa_K4592001 page on the iGEM Parts Registry) was input with PCC 7806 M. aeruginosa (the closest organism available on the database). In the generated library, the synthetic RBS with the highest predicted translation initiation rate was AAGGAGG with a translation initiation rate of 817373.40 au; this RBS is documented on the BBa_K4592003 page on the iGEM Parts Registry. We plan to use this synthetic RBS to express the eGFP reporter in UTEX 2385 M.aeruginosa with high efficiency, allowing us to validate transformations more easily. In doing so, we will also be able to empirically assess the strength of the synthetic RBS based on resulting eGFP fluorescence.

Table 1. Features of synthetic RBS (BBa_K4592003) predicted by Salis RBS Library Calculator v2.1.1.

Preparation of unmodified and modified pSPDY assembly via gene fragment amplification

gBlock Gene Fragments for the unmodified and modified pSPDY assemblies were amplified with flagged primers, introducing PaqC1 binding sites and fusion sites for Golden Gate assembly. To verify the success of this step, the resulting Golden Gate fragments were run on an agarose gel and compared to their expected sizes based on their in silico design. We verified the amplification of IDT gene blocks and addition of Golden Gate fusion sites of fragments with their expected amplicon band size. However, nonspecific amplification of gene fragments blue and ultraviolet was apparent. Amplification of the lime fragment, an assembly of the pSHDY backbone, yellow fragment, and green fragment, was not present on the gel, but is not necessary for assembly (Figure 5). For information on function and association of the color-coded Golden Gate fragments, see the section on plasmid construction in Engineering.

Figure 5. A 0.8% agarose gel depicting the PCR products of amplified IDT gene fragments used for golden gate assembly of unmodified and modified pSPDY plasmid. The gene fragments were amplified with added flag primers that inserted Golden Gate fusion sites during the PCR cycle.

Table 2.Gel content for fragment amplification verification and expected gene fragment sizes.

Testing the Chameleon Project

The unmodified pSPDY construct contains many restriction sites, while the modified pSPDY construct has been passed through Chameleon. Chameleon removed most of the putative restriction sites that were identified by the Stealth program through synonymous codon-optimization of protein-coding sequences. The sequences that are modified by Chameleon on both plasmids are depicted in red in Figures 6 and 7. All other sequences are not protein-coding, or are overlapping protein coding regions that could not be altered through synonymous codon-optimization. The Chameleon project will only consider non-overlapping protein-coding regions for restriction site avoidance to maintain the functionality of any plasmid. The Chameleon project only requires a partial genome sequence to optimize any plasmid sequence in GenBank format. This design functions as a generic tool for increasing the transformability of any GenBank plasmid, in any partially-sequenced species.

To validate the function of the Chameleon software, we annotated all of the putative restriction sites identified by the Stealth program on both modified and unmodified pSPDY. The sequences annotated in pink in Figures 6 and 7 were underrepresented in the partial genome we assembled for the UTEX 2385 M.aeruginosa> strain, and are therefore treated as putative restriction sites by the Chameleon project. The unmodified plasmid exhibited 197 total putative restriction sites, while the modified plasmid only exhibited 40. The Chameleon project successfully removed 76% of all palindromic, putative restriction sites identified by Stealth without altering any corresponding amino acid sequences (Fig. 8). These results demonstrate success of the Chameleon project bioinformatically.

Figure 6. Unmodified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the unmodified pSPDY plasmid. 197 putative restriction sites were identified on the unmodified pSPDY plasmid. Annotated regions colored in red were already passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

Figure 7. Modified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the modified pSPDY plasmid. After processing of the unmodified plasmid with Chameleon, the majority of restriction sites on coding regions were removed without altering corresponding amino acid sequences. 40 putative restriction sites were retained on the modified pSPDY plasmid. Annotated regions colored in red were passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

Figure 8. Comparison of chloramphenicol resistance gene (cat) on unmodified pSPDY (yellow) and modified pSPDY (red). Both versions of cat yield the same amino acid sequence despite different nucleotide sequences. There are 19 putative restriction sites identified on the unmodified cat CDS and 0 putative restriction sites identified on the modified cat CDS.

Future Plans

Due to time constraints, we were unable to complete much of the wet lab work that would validate our dry lab work. We are continuing to work on the full sequencing of the UTEX 2385 M.aeruginosa genome, validating assembly of both the modified and unmodified pSPDY plasmids, comparing transformation efficiency in UTEX 2385 M.aeruginosa between the two plasmids to provide empirical validation of our Chameleon software project, characterizing expression mediated by the CaMV35S core promoter in UTEX 2385 M.aeruginosa> , and empirically validating our synthetic RBS. During the wiki thaw and before the re-freeze, we will add updates on our progress on these goals

Beyond the initial scope of this iGEM project, we also intend to make use of the improvement in transformation efficiency in UTEX 2385 M.aeruginosa to make a high transformation efficiency plasmid to knock out expression of microcystin-synthesizing genes in M. aeruginosa, enabling us to effectively address its harmful algal blooms.

Considerations for Replication

If our experiments were to be replicated, we have several considerations to share with experimenters:

As both the Stealth program upon which our Chameleon project is built and the Chameleon project itself are continuing to be updated beyond this iGEM project, the outputs produced by the most recent versions may differ significantly from what is reported in our project even if the same inputs are used; experimenters may therefore consider using the static version of the Chameleon project hosted on the iGEM software Gitlab that corresponds to what was used for this project.
Our work in transforming both E. coli and UTEX 2385 M.aeruginosa with our plasmids was hampered by some of our early design decisions: namely, the use of chloramphenicol as a selection marker, the composition of our insert, and the sequences of our gBlock Gene Fragments.

Chloramphenicol presented challenges for the selection of transformants in that it is bacteriostatic rather than bacteriocidal; since M.aeruginosa> has a slow doubling time, it was difficult to distinguish between untransformed cells whose ribosomes were inhibited by chloramphenicol and transformed cells that were simply replicating slowly.
Our insert included a reporter (eGFP), but not the chloramphenicol resistance gene; this resistance gene was already on the pSHDY backbone, which meant that additional protocols were needed to determine whether observed transformants had taken up the intended pSPDY construct or pSHDY that had persisted through the PCR amplification and Golden Gate assembly. Experimenters should consider having different resistance genes on the backbone and insert such that transformants that took up the new plasmid can be separated from transformants that took up the old plasmid.
Our gBlock Gene Fragments were designed as the sequences that would exist in the assembled plasmid. As a result, flagged primers adding PaqC1 recognition sites and fusion sites to the PCR amplicon were required. These created unnecessary potential points of failure in that the flagged primers had more opportunities to dimerize or bind to off-target regions, yielding Golden Gate fragments that would lead to an incorrect plasmid assembly. Since the gBlock Gene Fragments are synthesized de novo, we advise experimenters to design the fragments to already include PaqC1 (or the preferred Type IIS restriction enzyme) recognition sites and fusion sites.

References

[1] M. Kaebernick, E. Dittmann, T. Börner, and B. A. Neilan, “Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial,” Appl. Environ. Microbiol., vol. 68, no. 2, pp. 449–455, Feb. 2002, doi: 10.1128/AEM.68.2.449-455.2002.

[2] A. C. Reis and H. M. Salis, “An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models,” ACS Synth. Biol., vol. 9, no. 11, pp. 3145–3156, Nov. 2020, doi: 10.1021/acssynbio.0c00394.

[3] I. Farasat, M. Kushwaha, J. Collens, M. Easterbrook, M. Guido, and H. M. Salis, “Efficient search, mapping, and optimization of multi‐protein genetic systems in diverse bacteria,” Mol. Syst. Biol., vol. 10, no. 6, p. 731, Jun. 2014, doi: 10.15252/msb.20134955.

[4] C. Y. Ng, I. Farasat, C. D. Maranas, and H. M. Salis, “Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration,” Metab. Eng., vol. 29, pp. 86–96, May 2015, doi: 10.1016/j.ymben.2015.03.001.