PHB

Understanding the Problem

Limitations of BC

Bacterial cellulose (BC) has excellent tensile strength and thermal stability, and is an attractive alternative to non-biodegradable synthetic packaging materials (1). Our project leverages BCs biocompatibility and non-toxicity to functionalize it with antimicrobial peptides. However, pure BC has limited elasticity, which makes it difficult to handle and ultimately limits its applications as food packaging (2).

This problem was affirmed by discussions with industry professionals. According to Christopher Clark and Chris Messent, two experts in produce sales and distribution, a key characteristic of packaging materials is their strength. During the transport from packaging facilities to grocery stores and retailers, produce packages must endure being stacked and exposed to moisture. It is also important that packaging materials maintain their structure after being exposed to transport conditions, as deteriorating packaging is unappealing to customers and reduces product marketability. This issue was highlighted by our discussions with John Kelly, the CEO of Food-to-Market. To produce a BC-based packaging material capable of meeting these needs, we needed to modify our biopolymer to increase its mechanical properties, without compromising its biodegradability or incorporating material from non-renewable sources. Our proposed solution? PHB.

Identifying PHB

How do we improve BC

Polyhydroxy-3-butyrate, or PHB, is a biodegradable plastic polymer produced in a wide range of bacteria under nutrient-limiting conditions. PHB forms as an intracellular lipid reserve material that accumulates as granules when the bacteria have access to external carbon, but lack nitrogen, phosphorus or oxygen (3). Natural PHB-producers include bacteria such as Bacillus spp., Nocardia spp., and Ralstonia eutropha (3).

Figure 1. PHB forms in the cytoplasmic space of bacterial cells. Adapted from SEM images by Obruca et al. (4)

The mechanical properties of PHB are comparable to those of conventional plastics used in produce packaging, such as polypropylene (PP) and polyethylene (PE) plastics (3). These polymers are stiff, highly hydrophobic, brittle and durable, which make them effective materials for protecting fruits and vegetables. However, while the recycling systems for PP and PE are expensive and process only a fraction of the plastics produced, PHB is entirely biodegradable (5).

Two halves make a whole solution

On its own, PHB has limited industrial applications due to its low thermal stability and high production costs (6,7). Pure PHB is very brittle and is vulnerable to breaking at low strain or melting at high temperatures (7). Moreover, the recovery of PHB from bacterial cells requires chemical or enzymatic digestion, and the product is generally pre-treated with chloroform before it can be blended with other polymers (7). These processes are generally expensive and complex.

However, in a co-culture with PHB-producing bacteria and K. xylinus, secreted PHB molecules can be incorporated into the bacterial cellulose (BC) fibres as they form, producing a matrix that is stronger than conventional BC (7). This approach has been shown to leverage the advantages of both polymers, and create a product that is less friable than pure BC, without the brittleness of PHB (6). By producing both polymers together, this method also eliminates the extra processing associated with producing and purifying PHB separately (7).

Figure 2. Adaptations of SEM images of (a) pure BC and (b) PHB pellets precipitated in BC. Adapted from Ding et al. (7)

Selecting organisms for co-culture

To eliminate the need to extract PHB from bacterial cells, we decided to use an engineered E. coli strain that not only produces PHB, but secretes it as well. Secreted PHB can be incorporated directly into BC without lysing the cells, which allows for the continuous production of PHB within the co-culture (8). This has important implications for lowering the cost of production.

The secretion system within PHB-producing E. coli relies on the production of phasin, an amphipathic protein which coats and stabilises PHB granules as they form in the bacterial cytoplasm (9). When bound, the phasin coating acts as a protective interphase between the highly hydrophobic PHB and the hydrophilic cytoplasm (9). Phasins also block interactions between individual PHB granules, and prevent them from aggregating into larger clusters that are too large to secrete (9). As such, phasin also became a focus for our proposed improved part design.

Our co-culture design includes PHB-producing E. coli and K. xylinus, our original BC-producing bacteria.

For more information about the co-culture method, please visit the co-culture subpage.

Part Design

Background of PHB secretion

The part design for this subproject builds directly on the work of other iGEM teams.

Previously, the Tokyo 2012 team developed a PHB-producing construct for the expression of PHB in E. coli (BBa_K934001). This operon codes for the three enzymes in the PHB biosynthesis pathway of a natural PHB-producing bacteria, Ralstonia Eutropha (Figure 3). The resulting engineered E. coli demonstrated successful intracellular PHB production.

Figure 3. PHB biosynthesis pathway in E. coli. (a) Glucose is metabolised into pyruvic acid by glycolysis. (b) Pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. (c) β-ketothiolase (coded in phaB) activity converts acetyl-CoA into acetoacetyl-CoA. (d) Acetoacetyl-Co-A is converted into R-3-hydroxybutyratyl-CoA by NADPH-dependent R-3-hydroxybutyratyl-CoA (coded in phaA). (e) R-3-hydroxybutyratyl-CoA is polymerized by PHA synthase (coded in phaC) to form PHB. (7)

Meanwhile, the Calgary 2017 team refined a phasin secretion system, whereby Hly-A tagged phasin is produced in E. coli, allowing for the secretion of PHB via the type I secretion pathway (10). In E. coli, the type 1 secretion system (T1SS) allows the single-step translocation of a large variety of proteins to the extracellular space (11). This team demonstrated the successful secretion of PHB, and their part (BBa_K934001) was the basis for our improved part.

Introducing our part design

Based on this previous work, our aim was to test if the strength of the ribosomal binding site (RBS) of the phasin coding sequence would have an effect on phasin expression levels. As phasin has an established role in PHB granule size, and the subsequent ability of the cell to secrete the PHB particles, our hope was that this proposed improvement could increase the concentration of PHB integrated into our BC product. Based on our mechanical tests, this could have implications on the strength and industrial applications of our BC biopolymer.

Our part design began with the sequence of the original Calgary 2017 phasin part (BBa_K934001), which includes a T7 promoter, a phasin coding sequence, and an HlyA sequence. First, we added a FLAG^TM tag upstream of the phasin-HlyA coding region to facilitate protein purification. The quantification of phasin expression levels will also provide important data from which to draw comparisons and conclusions once PHB production is achieved.

Next, we aimed to design four iterations of this part, each with a different RBS type upstream of the phasin coding region. As a control, one version contained B0034 (BBa_B0034), the RBS from the original part. The remaining three versions of the phasin part contained RBS types of varying relative strengths: B0030, B0035, and DeadRBS (a non-functional RBS to act as a negative control). The relative RBS strengths are described below (Table 1).

Table 1. List of relative RBS strengths

Rather than purchase four variations of the entire part, we decided to include them in primers, which, after PCR amplification, would be combined with the main phasin part described above. To integrate this part into the PHB-producing part (BBa_K934001), we also identified two sets of unique restriction enzyme cut sites within the coding region. Two versions of each primer pair were designed, one with each cut site set. Once amplified, this approach would yield a phasin part with each of the four RBS types (Figure 4).

Figure 4. (a) Schematic overview of the primer design approach used to amplify the FLAG-phasin-HlyA sequence with different RBS and RE types. (b) The four specific RBS types incorporated, for both restriction enzyme methods. The two ends are compatible with ligation in the Tokyo plasmid.

With the appropriate restriction enzyme cut sites in place, the integration of this part with the Tokyo 2012 PHB part requires a simple digestion and ligation.

For more information, please see the improved part and parts subpages.

Protein Expression and Uniaxial Testing

Mechanical properties

In order to quantify the effect of PHB on the mechanical properties of BC, uniaxial tensile tests were performed with both pure BC and a PHB-BC composite. The BC only samples were grown over four weeks at 30℃ in Hestrin-Schramm media with daily feeding. The PHB-BC composite samples were created by adding 1.5 mL of 0.25 g of PHB per mL of 100% acetic acid solution (0.25 g/mL) into BC palettes that have been growing for three weeks for seven days. After both samples had grown, they were autoclaved, purified using an NaHCO3 solution, and air dried. After drying, the samples were cut to size for uniaxial tests. Four replicates were produced of each polymer.

Figure 5. The ultimate tensile strength (MPa) of pure BC and composite PHB-BC.

Figure 6. The Young’s Modulus of pure BC and composite PHB-BC.

Figure 7. The maximum elongation of pure BC and composite PHB-BC.

Based on these results, BC showed an ultimate tensile strength 3.780MPa higher than PHB-BC (Figure 5). This value represents the maximum force the polymer can withstand before tearing. The Young’s Modulus of pure BC was also 758.2MPa higher than composite PHB-BC (Figure 6). This value indicates that pure BC is the stiffer of the two polymers, and is less flexible. Finally, the maximum elongation of composite PHB-BC was 0.02868 higher than pure BC (Figure 7). This value reflects the flexibility of both polymers when stretched.

Taken together, these uniaxial tests demonstrate that the introduction of PHB increases the flexibility of BC. This finding is significant, as the friability of pure BC was one of the principal concerns in its use as a potential packaging. Though there is a reduction in strength observed with the introduction of PHB into BC, this could be attributed to an uneven distribution of PHB in the BC matrix due to the ex situ method used to incorporate PHB. Furthermore, 100% glacial acetic acid has an approximate pH of 2.3 which was observed to cause a phenomenon called acid hydrolysis of the BC (12). Acid hydrolysis of cellulose is a classic way to break down cellulose into glucose and can be done using a concentrated acid (12). This may have also contributed to the less than optimal strength performance of the BC and PHB composite, as the BC itself has been broken down and weakened from the incorporation of PHB using a strong acid as a solvent (12). These findings further motivate the use of a co-culture approach to incorporate the PHB in situ as the BC fibres form. The production of smaller PHB particles through increased phasin expression may also foster a more even distribution.

RBS Strength on Regulating Phasin-HlyA Synthesis

A phasin-hlyA construct and primers that have the four different RBS sites (BBa_B0030, BBa_B0034, BBa_B0035, and DeadRBS or BBa_K4437500) and RE sites were created. There were two variations of the primers created with different cut sites based on the enzymes used, however it was evident that one of the cut sites between PstI and SpeI were not yielding successful digestion because the cut sites were too close to each other (~10 bp apart). Hence, the BstAPI, and BstBI RE and cut site primers were used to insert the complete phasin-hlyA construct with their respective RBS into the tokyo plasmid. The resultant constructs were: BBa_B0030-phasin-hlyA (BBa_K4437502), BBa_B0034-phasin-hlyA (BBa_K4437503), BBa_B0035-phasin-hlyA (BBa_K4437504), and DeadRBS-phasin-hlyA or BBa_K4437500-phasin-hlyA (BBa_K4437501).

A diagnostic digestion was performed, using PstI, which indicates that one band of length ~7100 bp should appear if the digestion and ligation was successful, while if the diglig was unsucessful than the bands would be a band size of 6400 bp.

Figure 8. Samples BBa_B0030-phasin-hlyA (BBa_K4437502) (2 and 5), BBa_B0034-phasin-hlyA (BBa_K4437503) (10, 11, 12), BBa_B0035-phasin-hlyA (BBa_K4437504) (16), and BBa_K4437500-phasin-hlyA (BBa_K4437501) (20) all had bands appear in the desired band size of ~7100 bp, indicating that for B0030, it was successfully digested, ligated, and transformed into TOP 10 cells, while for the other samples it was been transformed into BL21 cells. Due to time restrictions, the BBa_B0030-phasin-hlyA (BBa_K4437502) was not transformed into BL21 E.coli cells.

On the Western, there were three potential bands that could be observed because of hlyA can be cleaved from the phasin protein: phasin alone with a molecular mass of 20.960 kDa, hlyA alone with a molecular mass of 6.151 kDa, or a phasin-Hlya fusion protein with a molecular mass of 27.208 kDa.

Figure 9. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain autoinduced for 24 hours. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was stained with Coomassie blue. The gel was loaded as follows: (1) Ladder, (2) LanM positive control (BBa_K3945001), (3) BBa_K4437500-phasin-hlyA (BBa_K4437501), (4) BBa_B0034-phasin-hlyA (BBa_K4437503), (5) BBa_B0035-phasin-hlyA (BBa_K4437504), (6) Tokyo 2012 PHB construct only (BBa_K934001).

Another batch of transformed BL21 E. coli were autoinduced over 48 hours instead of 24 hours in hopes that there would be a more apparent difference in the amounts of phasin-hlyA produced between the different RBS. This is because swapping the RBS was intended for a fine-tuning effect. The cell lysate was used for an SDS to better compare the amount of phasin-hylA produced using anti-FLAG antibodies.

Figure 10. The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain autoinduced for 48 hours on an SDS page using Anti-FLAG antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was loaded as follows: (1) Ladder, (2) Tokyo 2012 PHB construct only (BBa_K934001) (3) BBa_K4437500-phasin-hlyA (BBa_K4437501), (4) BBa_B0034-phasin-hlyA (BBa_K4437503), (5) BBa_B0035-phasin-hlyA (BBa_K4437504).

The strongest RBS used, BBa_B0034 (BBa_K226002) had the most saturated and thickest band, indicating that phasin-hlyA translation was upregulated compared to the weaker RBS. The BBa_B0035 RBS, which is slightly weaker than the BBa_B0034 RBS showed slightly lower levels of phasin-hlyA synthesis, demonstrated by the lighter bands of both the cleaved and fusion phasin-hlyA protein. Lastly, the DeadRBS (BBa_K4437500) had levels of phasin-protein production near that of the negative Tokyo 2012 PHB (BBa_K934001) control. The slight bands in negative Tokyo 2012 PHB (BBa_K934001) control lane 2 can be attributed to leakage as its bands are similar to that of the DeadRBS-phasin lane (BBa_K4437500) which should have theoretically little to no protein synthesis.

Experiments in Progress

Experiments to assess the relationship between phasin expression and PHB secretion/production are currently underway. These include phasin and PHB production, purification and quantification. These experiments will culminate in further uniaxial tests to quantify the effect of different incorporation methods on BC and composite PHB-BC tensile strength.

Future Directions

Industrial Applications

Following the successful integration of PHB with BC, it is important to consider other applications within the packaging industry for this type of composite biopolymer. Conversations with key stakeholders -- from packaging experts to retail managers -- have highlighted the prevalence of nonrecyclable plastic used to protect and hold produce. The ability to finetune PHB concentrations in PHB-BC has implications for creating biodegradable packing materials with a diverse range of flexibility and mechanical properties. In the future, our team could also explore other properties relevant to fruit and vegetable packaging, including long-term durability, resistance to moisture over long periods of time, and the capacity for gas exchange.

References

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