Team:MADRID UCM/Experiments

Experiments - 4C_FUELS

Experimental design is key when planning a scientific project like ours. Ideas are great, but there has to be a viable way to test them. If not, they will remain just like that: ideas. In this page you will find the experiments we have carried out, as well as others that we planned but haven't got the chance to test.

Cell Encapsulation Experiments

An important ambit of 4C_Fuels project is to settle the foundations for the development of photobiocatalytic materials. During this year we have succesfully designed synthetesis protocols for the creation of various materials with photobiocatalytic potential.

In order to asses the potential of this materials for photobiocatalysis, we have evaluated its performance in terms of: Mechanical Properties, stability and cell viability, diffusional limitations and structural characterization.

Viability Test: Assesing Mechanical & Biological Stability

In order to obtain conclusions about the viability of the cells inside all materials a simple experiment was designed. This experiment consisted of synthetizing fresh materials all at once and keeping them with mild agitation at 28 ºC, 50 uE of illumination during an entire month. Tubes were open for 30 seconds without sterile conditions once every 5 days, in order to allow gas exchange and expose the materials to normal environmental conditions.

We have taken pictures of all the materials over the time in order to see how they are affected and how well they hold up. This way, we aim to either evaluate its mechanical and biological stability. The picture below shows the evolution of the different materials.


The Results

  • Beads As observed, the beads soon start to lose the green colour of the cyanobacteria within a few days. They turn from dark green to light green and finally to yellowish green.The approximate shelf life of the cyanobacteria (with live cyanobacteria) is around 4 days.

  • Thin films Although thin films make the cells stay alive longer than beads, they also lose their colour over the course of 5 days days, just like the former material. In addition, thin films are more easily contaminated than beads.

  • Foams In the case of foams, viability testing in culture medium or in water has not been done, as foams in liquid medium break down. They are very resistant and porous materials, until they are introduced in water or BG-11. In addition, they are fairly easily contaminated.

  • Silica gels Although the supernatant turns green when the silica gels are placed in BG-11, which is likely to indicate that the cyanobacteria have not encapsulated well; the gels hold up over the weeks, and the cells that are successfully encapsulated hold up well.

  • Yolk-shell Yolk-shell is one of the best performing materials. After weeks, they eventually lose their characteristic colour, a sign that the cells have been dying. Also included is a 40 minute video recorded in fast motion showing how the yolk-shell settles to the bottom of the Eppendorf. This behaviour is not characteristic of cells in culture medium, without encapsulation.
    yolk shell sedim
  • Silica gels with yolk-shell In contrast to gels with previously unencapsulated cells, the cells do not escape into the supernatant. Furthermore, the colour persists over the weeks, and the creation of bubbles by the cells during photosynthesis is observed even after a month before material synthesis.

Yolk Viability

Besides this results, we have also evaluated the yolk shell viability when kept in a buffer instead of growth media. We wanted to assess if the color retention of the material was just a sign of the stabilization of chlorophylls or an indication of encapsulated living cells. To do so, we aliquoted an equal amount of yolk-shell, centrifuged and resuspended it into BG-11 and also in PBS. They have been found to lose their colour in PBS in the priod of a week, while they retained their colour and continued producing gas bubbles, proof that the material can successfully keep cells alive for long periods of time.

The tube containing the BG-11 Yolk Shel suspension has been stored for more than 70 days without observing a decrease in Optical density or gas bubbling within the tube.

Diffusion characterization

We need to know the diffusional limit of each material, in order to conclude which will be the ideal material to allow nutrients, CO2 and the synthesized product to travel through the materials. In short, we wanted to test the mass transfer limitations of the synthesized materials.

Then, it is necessary that when cells that secrete n-butanol are encapsulated, the material in which they are introduced must be sufficiently porous, and with an adequate pore size to be able to allow the diffusion of the generated product outside the material.

Since a butanol producing strain was not yet available, experiments involving dyes diffusion are therefore considered. Thus, materials have been directly synthesized with the dyes. During the experiment the material has been placed in culture media with mild agitation and dye’s diffusion process has been studied.

Aliquots of the supernatant are taken and subsequently analysed in a spectrophotometer to see the increase in dye concentration in the supernatant for each material analysed. These experiments have been developed based on Lambert Beer's law, which relates absorbance to concentration, using spectrophotometric methods.

Experimental protocol:

In the case of each experiment, a concentrated 10% dye solution has been prepared, from which 0.2 mL has been mixed with 3 mL of precursor of each material. Then materials has been synthesized following the developed protocols and fixed amount of 0.4g of each material has been used for the diffusion experiments.

The Results

After performing the experiments, the diffusional limitation of each material could be observed attending to the relative release of the colorant to the media. However it is important to notice that with these assay we are evalueating the overall transpor limitation of an specific morphological and physichochemical configuration for each inmovilization system.

Materials like thin films will feature a higher surface-volume ratio, which eventually ease the release of the colorant to the media.

Graphic 1. BEADS with Congo red.

As seen in the graphic, the amount of dye diffused into the medium is increasing as time increases. At some points in time the absorbance is lower compared to the linear trend. This may be due to various factors, such as the sampling or the decrease of volume in the medium each time it is sampled.

Graphic 2. BEADS with violet crystal

Unlike the previous one, the trend of these data is closer to a logarithmic exponential trend. There are also some points that don’t follow this, but the tendency is clear. It is important to note that the data with respect to graph 1 are similar. So, it can be concluded that the diffusional limit is almost the same with both dyes.

Graphic 3. THIN FILMS with violet crystal in water and BG11

Although some points are not at the trend, the increasing absorbance with time is also clear. The absorbance is eight times the absorbance shown on the beads, so, we can conclude that thin films have a lower diffusional limit.

It is remarkable that there exists a difference between the thin films in water and in BG-11. We suspect that this could be caused by hardening effects on the thin film material when exposed to BG-11. In addition the change in ionic force caued by BG-11 may also alter the migration of employed colorants.

Graphic 4.SILICA GEL with Congo red

This is the graph with the trend that most closely approximates linearity. There is a steady increase in the absorbance of the samples, and it is concluded that their diffusional limit is lower than that of the beads and is like the thin films.

Attending to the observed results, the highest diffusion rates (in terms of absolute absorbance) has been obtained by the Thin Films materials, followed by the silica gels.

Morphological Characterization

In order to get an idea of the porosity and surface area of each material, as well as studying cell distribution and encapsulation success. The materials have been studied by SEM and optical microscopy. In the SEM it has been possible to visualise what the surfaces look like, and to some extent, the porosity of each one. It can be seen whether the material features a lamellar structure, fibrous or compact. Likewise is observable how in some materials cells are easily visible, while in other cell viability is severely compromised.

The following images corresponds with SEM and optical microscopy characterization of the synthesized materials.

Alginate Beads (No Freeze Dried)
Alginate Beads (Freeze Dried)
Alginate Beads Without Cyanobacteria (Freeze Dried)
Thin Film Without Cyanobacteria
Thin Film with cyanobacteria
Chitosan-Sepiolite Foam with cyanobacteria
Silica Gel with Cyanobacteria (Frezze-Dried)
Silica Gel with Cyanobacteria (No Frezze-Dried)
Silica Gel with Yolk-Shell (No Frezze-Dried)
Silica Gel with Yolk-Shell (No Frezze-Dried)
Suspension of Yolk-Shell Microstructures

Photobiocatalytic performance assays

Moving on from the previous characterization experiments, we decided to evaluate the performance of the photobiocatalysts in a real case scenario. To do so, we encapsulated a cyanobacteria strain able to secrete an easy-to-measure metabolite such as sucrose. We encapsulated exponentially growing cyanobacteria in Alginate-sepiolite beads, Yolk-Shell and Silica Gel Yolk-Shell materials, following the corresponding protocols. Biohybrid materials together with a non-encapsulated control were cultured in standard conditions.

During two weeks, supernatant samples were taken for each experimental sample. We recorded both OD720 and sucrose concentration, to then correlate sucrose production with biomass.

Our results showed that biohybrid materials did not compromise cyanobacteria growth. OD720 measures were taken for the course of two weeks for Yolk-Shells without reporting a decrease in growth. Their OD720 values remained constant at the initial value. For Alginate-sepiolite beads and Silica Gel Yolk-Shells OD measurements could not be taken due to the intrinsic material structure. However, they remained showing a vibrant green during the whole experiment.

Due to technical and timing issues, sucrose concentration could not be determined. Further research is needed to elucidate how each material affects metabolite secretion.

Metabolic engineering Experiments

n-Butanol Productivity Assays

We wanted to generate a butanol overproducing strain as a proof of concept for our phototrophic-based production platform. Sadly, due to cloning and strain transformation issues we ran out of time and no successful butanol-producing mutant cyanobacteria were obtained.

However, we planned ahead the experiments needed for the characterization of butanol production. In the following section we are going to briefly summarize our experimental design to asses n-butanol productivity performance.

Butanol is not an easy molecule to measure: it has no color and gives no visible phenotype to cells. Also, we couldn’t find cheap methods or kits to measure this simple alcohol. Then, a suitable way to assess n-butanol production was culturing cells in closed flasks with bicarbonate supplementation at 25mM. This allows us to avoid n-butanol vaporization from the media. Using a culture volume of 50 mL,, every day each bottle will be opened and a 1.5 mL sample would be removed, replacing the extracted volume by fresh BG-11 - HEPES media supplemented with 250 mM bicarbonate.

The extracted sample would be centrifuged to remove biomass and supernatant would be extracted with dichloromethane (DCM) for 20 minutes of intermitente shaking. Then centrifugation will be used again to recover a clean DCM supernatant, which will be eventually spiked with isobutanol as internal standard for HPLC determination. In order to study the n-butanol concentration, an HPCL equipment using a ionic exclusion column with 4 % to 8 % cross-linked sulfonated styrene-divinylbenzene (SDVB), like REZEX RFQ-Fast Acid H+ 8% column and a Flame Ionization Detector could be used.

As discussed in Metabolic Engineering we hypothesized that butanol biosynthesis could be improved by fine tuning pathways enzyme expression, by physically linking the two last enzymes of the biosynthesis pathway and by the introduction of synthetic bypasses to rewrite carbon utilization.

We designed multiple different constructs regarding these strategies. Our aim was to initially screen for the optimal performance mutant harboring a variant of n-butanol pathway alone. In this situation we aimed to compare the performance of the following strains.

  • Pathway re-arranged Strains: Where each protein is preceded by its own RBS and thus gets translated individually. They correspond with strains harboring BOH3_A, BOH3_B, BOH2AB, BOH1ABC construct variants, explained within the Genetic Design page.

  • Linked constructs GSG: where the stop codon of PduP has been suppressed and the RBS from Slr1192 has been replaced by a short-flexible linker comprising three amino acids: GSG. Corresponding with the strains harboring BOH1ABC-GSG and BOH2AB-GSG

  • Linked construct FR3F: similar as previous one but comprising a semi-rigid long linker: FRRRF (F-flexible GSG linker; R-rigid EAAAK linker). Corresponding with the strains harboring BOH1ABC-FR3F and BOH2AB-FR3F.

In the case of fusion enzynmes, we have previoysly carried out a bioinformatic analysis of the structure of the independent enzymes as well as both linker scenario strategies. No major conformational changes were found in the enzymes due to linker implementation. To test these three strategies, we would just measure butanol production of each construct and compare it with the rest of the obtained mutant strains.

Testing tolerance and secretion systems

Butanol is toxic to cells. One of its toxic effects is causing membrane instability, which affects phototrophic organisms to a greater extent due to their dependency on membranes (where the photosynthetic apparatus is located). That is the reason why we wanted to implement different strategies to improve the cell's tolerance to this alcohol.

Two proteins were chosen to be overexpressed in our modified cells: the chaperone HspA and the transcription factor SigB (see ‘Metabolic Engineering' to know more about them). Based on previous tolerance studies (refs.) we planned the following experiments to test whether their overexpression enhances butanol tolerance.

Cyanobacteria test strains and a wild-type control would be grown until 0.5 OD720. Then we would incubate our strains with butanol at different concentrations: 40 mg/L to resemble initial production concentrations, and 1.5 g/L as saturating concentration. Theoretically, after a 12h incubation period butanol has been able to diffuse and the correspondent butanol concentration must be present inside the cells. At this point we would compare growth by measuring OD720. Our hypothesis is that cyanobacteria that overexpress HspA, SigB, or both, would exhibit an improved tolerance and hence a higher growth compared with that of a wild type control.

These results haven’t been tested yet, but several studies have already seen this effect (refs.).

Another important tolerance strategy was the implementation of the butanol transporter AcrBv2 (see ‘Metabolic Engineering' to know more about it). Unlike HspA and SigB, this strategy doesn’t improve tolerance directly. AcrBv2 secretes butanol outside the cell, causing an indirect tolerance effect.

We would test AcrBv2 constructs with the same technique we would use for HspA and SigB, hypothesizing a similar trend of enhanced tolerance.

However, like other transporter proteins AcrBv2 is known to cause membrane stress if overexpressed without tight control. That is why we decided to include three differently regulated constructs when designing the AcrBv2 module, which we achieved by introducing different promoters upstream AcrBv2 gene (see ‘SynBio Design’ for further details):

  • Constitutive regulation with a well characterized promoter like J23100 or J23119. We expect AcrBv2 to cause a detrimental effect on growth due to membrane stress.

  • Inducible regulation with an inducible promoter like Pbad. In this case we would test different induction conditions in order to approximately get the optimal expression to obtain an improved tolerance but not an impaired growth.

  • Autoregulation based on membrane stress, using Pgntk promoter. With this promoter we would expect to get a similar effect to the optimal condition found with the inducible promoter, in a similar way as has previously found in available literature.

AcrBv2 is not only expected to improve tolerance, but also to remove butanol from the cell. Butanol secretion is a key feature of our project: it pushes the equilibrium of the biosynthetic pathway towards butanol production. This way, selecting the optimal expression of AcrBv2 is a crucial aspect to improve overall strain performance.

Once we get the best performing AcrBv2 construct we would address its secretion properties. From the same tolerance experiment, we would pellet the cells after the incubation period, lysate them and obtain the organic-soluble fraction. Then we would measure its butanol concentration. We expect to find a lower butanol concentration inside the cells that express AcrBv2 than in those of a wild type, due to AcrBv2-driven butanol secretion.

All the proposed hypotheses are based on results found in previous literature.

Evaluation of Gene Edition Systems

Besides designing a a metabolic engineering approach for cyanobacterial n-butanol production, we also found the need for efficient tools for cyanobacterial genome edition. Because of this, we decided to develop resources which could help us to speed up our strain engineering workflow and become an useful tool for the synthetic biology community.

In our team we have created NSFinder, a program that looks for putative neutral sites within a given genome (see Software to know how this works). We planned an experimental strategy to test how NSFinder would perform in selecting novel neutral sites in cyanobacteria. Also, this system is thought to work with new recombineering techniques like the I-SceI-based counter-selection strategy we implemented (see Engineering for detailed information).

Evaluation of Neutral Sites and I-SceI Unmarking System

Thanks to pJAY plasmids (see Genetic Design) we would assess each identified putative neutral site through YFP expression. However, with these experiments we would also check for the efficiency of I-SceI-mediated integration in phototrophic microorganisms, something that, to our knowledge, has not been verified to date (see Engineering for further details). Taking these together, the following experimental design had two objectives:

  • Address whether predicted neutral sites are suitable for genomic integration, and evaluate its relative expression levels.

  • Evaluate the efficiency of our engineering design as a genome integration tool, compared with traditional approaches.

  • Compare the behavior of marked and unmarked strains when using different neutral sites.

NSFinder found more than 100 putative neutral sites in PCC11801’s genome. Among all of these, we selected four for their characterization. Thus, we would construct one pJAY plasmid for each of them. These plasmids would be integrated in the genome selecting for single recombination events. This process would create antibiotic resistant mutants. Afterwards, I-SceI expression plasmid would be introduced.

Once I-SceI cuts in their target regions, there is a 50% chance of getting unmarked mutants or a reverted wild type, depending on the way the homologous recombination is performed for the double strand break repair. As cyanobacteria generally have multiple copies of their chromosome, PCR checking is needed along the way to confirm complete segregation of unmarked mutants.

In the end, we would have four pJAY strains, each of them harboring a YFP expression cassette in a different putative neutral site of the genome. By carrying out fluorescence measurements of each of the strains we would be able to characterize the potential of either neutral site as an expression location within PCC11801’s genome.

Also, an I-SceI expression plasmid would be built, in order to express the I-SceI restriction enzyme. Once modified acceptor plasmids are integrated in the genome, I-SceI would cut DNA at its recognition sequence causing double-strand breaks. Repairing mechanisms would then drive recombination , leading to antibiotic counterselection, leaving an antibiotic-free insertion in the microorganism’s genome.

By using I-SceI counterselection strategy, we rely on single recombination events that would introduce the whole pJAY plasmid into the genome. Single recombinants might express two antibiotic resistances: the insert resistance together with the backbone resistance. In this way we could select single recombinants by exposure to both antibiotics. Also, colonies should be expressing YFP.

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