Team:MiamiU OH/Engineering

iGEM 2021 | Miami University

Engineering Success

Our goal here at CROP is to revolutionize the future of food production to meet the demands of a growing human population. Through successful use of the engineering cycle, we…

  • Designed synthetic pathways that enable a more robust carbon regeneration process during carbon fixation
  • Built and integrated our system into the model cyanobacteria Synechococcus elongatus PCC 7942
  • Tested our pathway(s) in vitro through computer modeling and in vivo through growth experiments
  • Learned about experimental mistakes, repeated testing, and were able to learn about the effect of our synthetic pathways on cyanobacterial growth. We also researched and discussed the possible reasons behind our results, and proposed future experiments to further optimize our design

We were able to generate successful transformants with all of our plasmids, with varying degrees of segregation, producing noticeable cell activity change detailed on our Proof of Concept page. This demonstrates the viability of our parts, detailed on the Contributions page.

We submitted parts(BBa_K4047034) and (BBa_K4047016) as our best characterized new parts, respectively encoding transaldolase and gentamicin resistance.

Design

Pathway Design

We began our summer by researching the limitations of photosynthesis to narrow down where it would be most effective for us to reengineer. Two of our team members, Hope Townsend and Avery Imes, were able to discuss initial ideas with our primary investigator (PI) Dr. Xin Wang, an expert in the synthetic biology field. This provided the necessary guidance for further research once the team was established.

Our first objective was to narrow down what part of photosynthesis we wanted to focus on, as it is a complex 170-step process with many areas for improvement (1). One consideration was the light reactions. Oftentimes at excessive light levels, a plant’s light-capturing-machinery becomes saturated, having destructive consequences (2). While this is an important limit to photosynthetic efficiency, work on the complex light-based structures would have required more resources and previous knowledge than our team could reasonably acquire within the competition timeframe . In addition, if later steps of photosynthesis can’t “keep up” with the energy flow coming in from light capture, then the process can become stalled and thus no overall improvement is gained. From this perspective, it was ideal to focus on energy and metabolite allocation in downstream photosynthetic steps to optimize their constant flow and renewal.

From there we looked to the Calvin-Benson-Bassham (CBB) cycle, which comprises the second portion of photosynthesis. The CBB cycle can be further subdivided into three parts: carbon fixation, reduction, and regeneration. The fixation step is catalyzed by the notoriously inefficient enzyme RubisCO. Decades of research have attempted to improve RubisCO without any significant breakthroughs (3). Through billions of years of evolutionary selection, Rubisco activity is still slow and promiscuous, which leads to the photorespiration process, another metabolic process that lowers photosynthetic efficiency. There is also a theory in the field that Rubisco has already been optimized through evolution due to its central metabolic role in photosynthetic organisms (4). Thus there is not much opportunity left for its activity to be further improved.

During the reduction portion, the conversion of 3-phosphoglycerate to glyceraldehyde-3-phosphate is simple and relatively efficient with little room for obvious improvement. This led us to focus on regeneration; this step regenerates a necessary intermediate of the CBB cycle, ribulose-1,5-bisphosphate (RuBP), through a series of reactions (5). This series of reactions produces intermediates that are often consumed by reactions not involved in the CBB cycle, taking resources away from direct RuBP formation. We decided that we would find a more direct way of converting the 3 carbon compound glyceraldehyde-3-phosphate into the 5 carbon compound ribulose-1,5- bisphosphate.

Our literature searches led us to the design of several potential alternative carbon regeneration pathways, two of which, the transaldolase and the glycolaldehyde pathways, showed the most promise. The glycolaldehyde pathway was revised several times until the final version was created, as it was the most novel and required the most extensive analysis for viability. Both pathways were tested using computer modeling. Due to time and resource restrictions, we chose to perform in vivo experiments using only the transaldolase pathway.

Build

Constructing the Plasmids

Our primers were carefully designed using NEBuilder. This process was in itself an iterative cycle, in which the student team members designed the primers on their own and then presented them to our PI. We were then able to receive commentary on issues such as length, annealing temperature, and more that allowed for more efficient redesign.

Once the proper primers were ordered, we performed PCR to get the desired fragments for Gibson Assembly. We amplified four components for the overexpression enzymes which were pT_tal, pF_fbp, pTF_tal, and pTF_fbp. Amplification was performed using a standard PCR protocol, using the high-fidelity Q5 polymerase to ensure the fewest number of introduced mutations. These fragments contained the genome sequence for transaldolase (tal) or fructose-1,6-bisphosphatase (fbp) as well as overlapping sequences for gibson assembly and restriction enzyme sites for confirmation testing. We used these fragments to assemble three different plasmid vectors to overexpress the enzymes of interest - pGEM-tal (BBa_K4047036), pGEM-fbp (BBa_K4047037), and pGEM-tal+fbp (BBa_K4047038). Before assembly we had to perform a restriction digest to isolate the backbone of pAM2991 (BBa_K4047024). This backbone provides the spectinomycin/streptomycin resistance for selection of successful transformants. It also provides the lacI gene, lacIq promoter, trc promoter, and lac operator. The lac repressor LacI, encoded by the lacI gene, binds to the lac operator to inhibit transcription. We used this regulatory system to induce higher gene expression of tal, fbp, or both through addition of IPTG during growth experiments.

We also amplified (via PCR) four components for the sedoheptulose -1,7- bisphosphatase (SBPase) deletion vector- a kanamycin resistance (kmR) cassette (BBa_K4047019), a backbone from a previously established plasmid pXWK3-glgC (BBa_K4047018), an upstream fragment of the SBPase gene glpX (BBa_K4047022), and a downstream fragment of glpX (BBa_K4047023). We gibson assembled the components to create the deletion plasmid pGEM-SBPase (BBa_K4047039), enabling the interruption of glpX to prevent SBPase expression with a kanamycin resistance cassette. The maintenance of the upstream and downstream portions of glpX ensures enough homology for proper crossover for successful gene deletion. The backbone amplified from pXWK3-glgC contained the gentamicin resistance (gmR) cassette for distinguishing between single and double crossovers. This was important because a single recombination event integrates the entire plasmid and results in a duplication of the glpX- one wildtype allele and one mutant allele (6). For these single crossovers, transformed cells would have both gmR and kmR, and thus grow on both kanamycin and gentamicin-containing plates. Only a double crossover results in the desired deletion mutant (6). In this double crossover, only the kmR cassette is inserted between the targeted upstream and downstream glpX fragments, inserting the antibiotic into glpX and preventing the synthesis of functional SBPase. The gmR antibiotic, in the plasmid backbone, is not acquired. We could, therefore, select colonies that only grew on media containing kanamycin and not gentamicin to ensure a proper deletion as well as removal of the introduced plasmid vector and wild type copy of glpX.

Transforming and Confirming Colonies

Once our three overexpression and one deletion plasmid were assembled, we used them to transform Escherichia coli DH10β. This was necessary to get a large set of copies of the plasmid for proper transformation into our organism of interest, S. elongatus PCC 7942. Once we replicated and purified the plasmids from E. coli, we confirmed the plasmid components via restriction enzyme digestion, and performed transformation in S. elongatus PCC 7942. For our deletion colonies, we used km and gm selection to locate double crossovers and segregation/colony PCR to evaluate the extent of plasmid integration. S. elongatus PCC 7942 has multiple copies of its genome and not all copies may contain our desired insert. This latter process was similarly performed for our overexpression colonies. We also used several rounds of segregation PCR to monitor the success of integration of our genes with IPTG activated promoters to allow for overexpression at neutral site I (NSI).

Test

Initially, our testing was delayed at the transformation and segregation stage, detailed below in the learning section. We were able to repeat our transformations with new stock colonies and proceed to viable testing detailed here.

We were able to achieve a double crossover colony for glpX deletion. However, after several rounds of segregation PCR, we concluded that complete segregation with no remaining functional SBPase was difficult to achieve and not within the competition timeframe (see Notebook). In other words, only some of the genomic copies in the cells had the successful deletion while the remaining copies maintained wild type. This is understandable because glpX, encoding SBPase, is considered an essential gene, so only very few copies could have the deletion without affecting the viability of the cell (7).

Photo of testing. Figure 1. km5+ from 8/10 with original PCC 7942 colonies transformed with pGEM-SBPase.

Photo of testing. Figure 2. km5+ from 8/23 with original colonies in quadrants 1-10 (with several transfers taken afterwards).

Photo of testing. Figure 3. gm2+ from 8/23 with original colonies in quadrants 1-10 (with several transfers taken afterwards).

Photo of testing. Figure 4. km5+ from 9/9 with dilution series of colony #3 (km5+ 8/30) on quadrants 1-11.

We were additionally able to achieve partial segregation for three tal, three fbp, and four tal-fbp colonies. We compared growth of the wild type strain and the three tal, three fbp, and three tal-fbp colonies using growth curves meant to replicate day-night cycles. Cells alternated between 10 hour light and 14 hour dark cycles at 30ºC for a total of 5 days to replicate the usual stress of alternating days and nights.

Our growth experiment showed that overexpressing transaldolase or FBPase alone (Figure 5) leads to less growth compared to the wildtype strain. We attribute these results to metabolic stress caused by an accumulation of F6P from FBPase and transaldolase forward activity pulling intermediates away from carbon regeneration into the pentose phosphate pathway. Yet when transaldolase and FBPase were overexpressed together, rather than growing less robustly as seen for individual gene overexpression strains, cells picked up growth faster during the light phase and reached a higher yield than with either transaldolase or FBPase alone. The growth profile supported our modeling results; our transaldolase pathway can be favored over the native CBB regeneration when FBPase provides hexose phosphates. Without a deletion of SBPase, our pathway had to compete with the native pathway, yet was still able to maintain growth similar to WT in the first few light/dark cycles (Figure 6). In conclusion, our results show that our proposed transaldolase pathway is not only functional but is favored by the cell when hexose phosphates are more available than triose phosphates. By deleting SBPase and excluding the native pathway, we expect to see growth that matches or exceeds that of wildtype. Further details of these results and analysis can be found in our Proof of Concept page. Growth curves for tal, fbp, and tal-fbp overexpression cells. Figure 5. Growth curves for tal, fbp, and tal-fbp overexpression cells. Growth curves for wild type and tal-fbp overexpression cells. Figure 6. Growth curves for wild type and tal-fbp overexpression cells.

Growth curves for tal, fbp, and tal-fbp overexpression cells Figure 7: Growth curves for tal, fbp, and tal-fbp overexpression cells.

Growth curves for wild type and tal-fbp overexpression cells Figure 8: Growth curves for wild type and tal-fbp overexpression cells.

Learn and Design 2.0

After several repeats of transformations and segregation PCR, we learned to troubleshoot and accept the natural struggles of working with biological systems. For several initial rounds of transformation, the growth of transformants was relatively dense. Generally, efficiency of transformation is limited; when growth on the agar plates resembled a “lawn” rather than individual colonies, we had to consider 1) if there antibiotic was present on the plate and 2) if antibiotic resistance was introduced from somewhere other than our plasmid (i.e. a previously transformed strain contaminated our wildtype cultures). To address the first possibility, we remade agar plates and delayed antibiotic addition to when the temperature of the agar wouldn’t denature it. As there was still lawn-like growth, we revived stored wildtype colonies and used those for our next transformation. These transformants showed the expected thinner density and thus, we concluded the wildtype stock previously used was contaminated. We have reason to believe this was due to contaminated media used to supplement the colonies, which must be monitored in the future.

Because contamination was the likely cause of our initial problems, we also encountered the importance of careful sterilization and antiseptic technique. Not only does it maintain result value, but it also saves time from repeating experiments and reviving new colonies. S. elongatus PCC 7942 is a slower growing organism than E. coli and transformants can even take up to a week to appear.

Although we only completed one full iteration of the engineering process, we learned to navigate experimental issues and repeated several elements of the steps within the cycle. We were able to successfully produce transformants. We registered all parts used, and although several of our linked parts do not have BioBrick compatibility due to the enzymes available to our lab, the basic parts comprising them have a variety of compatibility viable for future iGEM teams.

In the future, we plan to test whether or not we can get a full deletion of SBPase in our overexpression strains. We expect that our proposed alternative carbon regeneration cycles would circumvent the need for SBPase and therefore full deletion would be more achievable.

References

1. Modeling Photosynthesis. RIPE. (https://ripe.illinois.edu/objectives/modeling-photosynthesis)

2. Leister D. 2012. How Can the Light Reactions of Photosynthesis be Improved in Plants? Front Plant Sci 3:199. (https://doi.org/10.3389/fpls.2012.00199)

3. Whitney SM, Sharwood RE, Orr D, White SJ, Alonso H, Galmés J. 2011. Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria. PNAS 108:14688–14693. (https://doi.org/10.1073/pnas.1109503108)

4. Bar-Even A, Flamholz A, Noor E, Milo R. 2012. Thermodynamic constraints shape the structure of carbon fixation pathways. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817:1646–1659. (https://doi.org/10.1016/j.bbabio.2012.05.002)

5. Raines CA. 2003. The Calvin cycle revisited. Photosynthesis Research 75:1–10. (https://doi.org/10.1023/A:1022421515027)

6. Clerico EM, Ditty JL, Golden SS. 2007. Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria, p. 155–171. In Rosato, E (ed.), Circadian Rhythms: Methods and Protocols. Humana Press, Totowa, NJ. (https://doi.org/10.1007/978-1-59745-257-1_11)

7. Tamoi M, Takeda T, Shigeoka S. 1999. Functional Analysis of Fructose-1,6-Bisphosphatase Isozymes (fbp-I and fbp-II Gene Products) in Cyanobacteria. Plant and Cell Physiology 40:257–261. (https://doi.org/10.1093/oxfordjournals.pcp.a029536)

Explore Next

A close up of gears.

Parts

See our full list of parts entered into the iGEM Registry.

A close up of cyanobacteria.

Proof of Concept

Explore the experiments conducted by our wet lab team and see the results of our genetic modification in cyanobacteria!